2-benzoylaminobenzamide derivatives as Bcl-3 inhibitors

ABSTRACT

The invention relates to a compound of general formula (I): 
                         
wherein R 1 , R 2 , R 3 , R 4 , Q, m and n are as defined herein. The compounds are inhibitors of Bcl3 and are useful for the treatment of cancer, particularly metastatic cancer.

RELATED APPLICATIONS

This application is a U.S. National Phase application, filed under 35U.S.C. § 371, of International Application No. PCT/EP2014/066564, filedJul. 31, 2014, which claims priority to, and the benefit of, GreatBritain Application No. 1313664.3, filed Jul. 31, 2013, the entirecontents of each of which are incorporated herein by reference in theirentireties.

FIELD OF THE INVENTION

The invention relates to novel inhibitors of B-cell Lymphoma 3 (Bcl-3);novel therapeutics or compositions comprising said inhibitors of B-cellLymphoma 3 (Bcl-3); and the use of said therapeutics or compositions totreat cancer and particularly metastatic cancer or secondary cancers.

BACKGROUND OF THE INVENTION

Cancer is a broad group of multiple diseases, all involving unregulatedcell growth. In cancer, cells divide and grow uncontrollably, formingmalignant tumours. In 2008 approximately 12.7 million cancers werediagnosed and 7.6 million people died of cancer worldwide. Cancers as agroup account for approximately 13% of all deaths each year with themost common being: lung cancer (1.4 million deaths), stomach cancer(740,000 deaths), liver cancer (700,000 deaths), colorectal cancer(610,000 deaths), and breast cancer (460,000 deaths). This makesinvasive cancer the leading cause of death in the developed world andthe second leading cause of death in the developing world.

There are more than 100 different types of cancer, the name for each isderived from the tissue or organ from which it originates. Determiningthe causes of different cancer types is a complex process as it isgenerally acknowledged that cancer formation is a multi-faceted process.Cancers are primarily an environmental disease with 90-95% of casesattributed to environmental factors and 5-10% due to genetics. Thechances of surviving the disease vary greatly according to the type andlocation of the cancer, and the extent of disease at the commencement oftreatment.

Metastasis, or metastatic disease, is the spread of a cancer from anoriginating tissue or organ to another tissue or organ. The cells whichconstitute the primary cancerous tumour commonly undergo metaplasia,followed by dysplasia and then anaplasia, resulting in a malignantphenotype. This malignant phenotype allows for intravasation into thecirculation, followed by extravasation to a second site fortumourigenesis. After the tumour cells have migrated to another site,they re-penetrate the vessel or walls and continue to multiply,eventually forming another clinically detectable tumour (secondarytumours). Whilst treatment regimens and therapies for primary tumoursare much better understood, with improved efficacy and success rates,and whilst some types of metastatic cancer can be cured with suchcurrent treatments, most metastatic cancers show poor response.Treatments for metastatic disease do exist, such as systemic therapy(chemotherapy, biological therapy, targeted therapy, hormonal therapy),local therapy (surgery, radiation therapy), or a combination of thesetreatments. However, most often the primary goal of these treatments isto control the growth of the cancer or to relieve symptoms caused bysame. It is therefore generally considered that most people who die ofcancer die of metastatic disease.

For example, breast cancer is the most common cancer in the UK and themost prevalent cancer in women worldwide (there were 1.38 million newcases diagnosed worldwide in 2008 accounting for 23% of all new cancercases). The relative survival of breast cancer patients has increaseddramatically over the last 35 years, with localised disease largelyconsidered to be curable, however, up to 20% of patients are likely todevelop metastatic disease which has poor prognosis. Breast cancertumours show distinct and reproducible subtypes of breast carcinomaassociated with different clinical outcomes. ERBB (Her2)-positive breastcancers, which constitute around one third of all breast tumours, have aparticularly poor prognosis, exhibiting resistance to first lineanti-cancer drugs, and frequently developing metastatic disease—the mostcommon cause of patient death. This particular clinical subtype istherefore an aggressive form of breast cancer with increased incidenceof metastasis and consequently poor prognosis.

Therefore improved understanding of cancer progression towardsaggressive metastatic forms and tumour cell-specific molecular pathwaysis necessary to improve and lead to new therapies. However, there arevarious difficulties associated with targeting metastases anddiscovering novel molecular targets. The differences between the earlystages and metastases development require novel targeted therapy thatwill differ from targeted therapy for primary tumours. Moreover, thetarget gene has to be detectable in the disseminated tumour cells or inthe primary tumour before metastases. Due to the potential latencyperiod between primary tumour development and metastatic disease,targeted therapy requires administration for prolonged periods withfewer side effects than conventional cancer therapies.

NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells)is a protein complex that controls the transcription of DNA, and isinvolved in cellular responses to stimuli such as stress, cytokines,free radicals, ultraviolet irradiation, oxidized LDL, and bacterial orviral antigens. Members of the NF-κB family can both induce and repressgene expression through binding to DNA sequences, and regulate numerousgenes that control programmed cell death, cell adhesion, proliferation,immunity and inflammation.

A connection between inflammation and carcinogenesis has been known fora long time, and it is therefore known that NF-κB provides a linkbetween inflammation and cancer progression. Further, NF-κB is widelyused by eukaryotic cells as a regulator of genes that control cellproliferation and cell survival. As such, many different types of humantumours have deregulated NF-κB: that is, NF-κB is constitutively active.Deregulated NF-κB has been documented in many cancers, including solidcancers such as breast, melanoma, lung, colon, pancreatic, oesophageal,and also haematological malignancies. For example, it has been shownthat increased NF-κB activation was evident in 86% of HER2+/ER− breastcancers and in 33% of basal like cancers, which are associated with ashortened disease-free interval, poor survival and resistance to cancertherapy (1). Moreover, NF-κB activation in tumour cells,tumour-associated stromal and endothelial cells is thought to play arole in tumour progression and invasion (2).

In tumour cells, NF-κB is active either due to mutations in genesencoding the NF-κB transcription factors themselves or in genes thatcontrol NF-κB activity (such as IκB genes); in addition, some tumourcells secrete factors that cause NF-κB to become active. Blocking NF-κBcan cause tumour cells to stop proliferating, to die, or to become moresensitive to the action of anti-tumour agents. Thus, NF-κB is thesubject of much active research among pharmaceutical companies as atarget for anti-cancer therapy, and numerous inhibitors of NF-κB andinducers of NF-κB are available.

B-cell Lymphoma 3 (Bcl-3) is a proto-oncogene modulating NF-κBsignalling, which was first identified as a chromosome translocation inB-cell chronic lymphocytic leukaemia. Deregulated Bcl-3 over-expressionhas been reported in numerous tumours including several leukaemias andlymphomas, such as anaplastic large cell lymphomas (ALCLs), classicHodgkin lymphomas (cHL) and non-Hodgkin's lymphoma (3&4). Additionally,deregulated expression has also been observed in solid tumour cancers,such as breast cancer, nasopharyngeal carcinoma, and hepatocarcinomas(5-7).

A role for NF-κB and Bcl-3 in metastatic colorectal cancer has also beenshown, where it was observed that NF-κB activation occurs prior tometastatic spread (8). Notably, Bcl-3 expression was also observed innormal and tumour tissue, but a correlation between nuclear Bcl-3 andpatient survival was observed. Bcl-3 expression has also been found tobe increased in breast cancer cell lines and patient breast cancersamples versus non-tumorigenic cell lines and normal adjacent tissue,respectively (9). Cells overexpressing Bcl-3 also resulted in asignificantly higher number of tumours which supports the role for Bcl-3in breast cancer progression (10).

The underlying oncogenic function of Bcl-3 has never been fullyelucidated. However, established thinking based on experiments performedon cancer cell lines in vitro is that it has a role in increasedcellular proliferation and cell survival.

In contrast, we have previously shown that Bcl-3 specifically promotesthe formation of metastasis of ErbB2 breast cancer driven tumours (11).Although primary tumour growth in the Bcl-3 deficient ErbB2 (MMTV/neu)murine model was not affected, it was shown that the occurrence ofdeveloped lung metastasis from a primary breast tumour was significantlyreduced by 40%. Moreover, a significant reduction in mitotic index andapoptosis was observed in secondary tumour lesions but not in primarytumours. Furthermore, through gene expression knock down studies, it wasshown that deletion of Bcl-3 resulted in an 80% decrease in lungmetastases, which was attributed to loss of cell migration butimportantly with no effect upon normal mammary function or overallsystemic viability. The implication from these observations, andsupported by leading thinkers in the field, is that specific targetingof individual NF-κB subunits or their co-activators may be a morebeneficial therapeutic strategy than suppressing their upstreamregulators which appear to exhibit detrimental systemic toxicity. Thistherefore suggests Bcl-3 may represent a suitable therapeutic target forpreventing cancer metastasis and secondary tumour formation.

Bcl-3 modulates transcription through binding to the proteins p50 andp52 from the NF-κB family. We have found that Bcl-3 function can beinhibited by disruption of this binding and that Bcl-3 suppressionresults in a decrease in NF-κB activation, cell migration andproliferative capacity. Using molecular modelling of the Bcl3 proteinbound to its cognate NF-κB protein partners, we have identified a novelpharmacophore on the Bcl3 protein, which influences its interaction withthe NF-κB proteins.

We have identified compounds which are capable of suppressing Bcl3-NF-κBprotein interactions, inhibiting NF-κB signalling and attenuating thecellular characteristics contributing to the metastatic phenotypeobserved in vivo. These compounds are therefore useful for the treatmentor prevention of cancer, especially metastatic disease and secondarytumour formation.

STATEMENTS OF INVENTION

According to a first aspect of the invention there is provided acompound of general formula (I):

or a salt thereof, wherein:A is phenyl or pyridyl;each R¹ is independently hydrogen, halo, nitro, cyano, C₁₋₆ alkyl, C₂₋₆alkenyl, C₁₋₆ haloalkyl, OR⁵ or N(R⁵R⁶);

-   -   each of R⁵ and R⁶ is independently hydrogen, C₁₋₆ alkyl or C₁₋₆        haloalkyl or, where two OR⁵ substituents are attached to        adjacent carbon atoms they may combine with the carbon atoms to        which they are attached to form a 5- or 6-membered ring;        m is 1-5;        R² is hydrogen or C₁₋₆ alkyl;        Q is a bivalent linker comprising a C₁₋₆ alkylene group in which        one or two —CH₂—moieties are optionally replaced by —O— or in        which two hydrogen atoms are optionally replaced with a group        (CH₂)_(p), wherein p is 2-4, such that a cyclic group is formed,        wherein the bivalent linker may optionally be substituted with        one or more substituents selected from halo and OH;        R³ is phenyl or a 5- or 6-membered heteroaryl group either of        which may optionally be substituted with one or more        substituents selected from C₁₋₄ alkyl, C₁₋₄ haloalkyl, or OH; or        R³ is NR⁷R⁸;    -   wherein each of R⁷ and R⁸ is independently hydrogen or C₁₋₆        alkyl or R⁷ and R⁸ together with the nitrogen atom to which they        are attached may form a 5 to 7 membered heterocyclic ring        optionally containing one or more other heteroatoms selected        from N, O and S;        each R⁴ is independently hydrogen, halo, nitro, cyano, C₁₋₆        alkyl, C₂₋₆ alkenyl, C₁₋₆ haloalkyl, OR⁹ or N(R⁹R¹⁰); and        each of R⁹ and R¹⁰ is independently hydrogen, C₁₋₆ alkyl or C₁₋₆        haloalkyl or, where two OR⁹ substituents are attached to        adjacent carbon atoms they may combine with the carbon atoms to        which they are attached to form a 5- or 6-membered heterocyclic        ring;        n is 1-4,        provided that the compound is not        2-[(2-fluorobenzoyl)amino-N-2-morpholin-4-ylethyl)benzamide.

In a further aspect of the invention there is provided a compound ofgeneral formula (I) include compounds of general formula (Ia):

or a salt thereof, wherein:each R¹ is independently halo, nitro, cyano, C₁₋₆ alkyl, C₂₋₆ alkenyl,C₁₋₆ haloalkyl, OR⁵ or N(R⁵R⁶);

-   -   each of R⁵ and R⁶ is independently hydrogen, C₁₋₆ alkyl or C₁₋₆        haloalkyl;        m is 0, 1 or 2;        Q is (CH₂)_(p);        p is 1, 2, 3 or 4;        R³ is morpholinyl, piperidinyl, piperazinyl, pyrrolidinyl,        pyrazolidinyl, imidazolinyl, pyridyl, pyrrolyl, pyrimidinyl,        imdizolyl, triazolyl or amino;        each R⁴ is independently nitro, cyano, C₁₋₆ alkyl, C₂₋₆ alkenyl,        C₁₋₆ haloalkyl, OR⁹ or N(R⁹R¹⁰);    -   each of R⁹ and R¹⁰ is independently hydrogen, C₁₋₆ alkyl or C₁₋₆        haloalkyl; and        n is 0, 1 or 2,        provided that the compound is not        2-[(2-fluorobenzoyl)amino-N-2-morpholin-4-ylethyl)benzamide.

As discussed above, these compounds are capable of suppressingBcl3-NF-κB protein interactions, inhibiting NF-κB NF-κB signalling andattenuating the cellular characteristics contributing to the metastaticphenotype observed in vivo and therefore the compounds are suitable forthe treatment of cancer, especially for the treatment or prevention ofmetastatic cancer or secondary tumours.

Throughout the description and claims of this specification, the words“comprise” and “contain” and variations of the words, for example“comprising” and “comprises”, mean “including but not limited to” and donot exclude other moieties, additives, components, integers or steps.Throughout the description and claims of this specification, thesingular encompasses the plural unless the context otherwise requires.In particular, where the indefinite article is used, the specificationis to be understood as contemplating plurality as well as singularity,unless the context requires otherwise.

All references, including any patent or patent application, cited inthis specification are hereby incorporated by reference. No admission ismade that any reference constitutes prior art. Further, no admission ismade that any of the prior art constitutes part of the common generalknowledge in the art.

Preferred features of each aspect of the invention may be as describedin connection with any of the other aspects.

In the present specification, references to compounds of general formula(I) or (Ia) includes amorphous and crystalline forms, including allpolymorphs, as well as isotopic variants, for example compounds offormula (I) or (Ia) in which one or more hydrogen atoms is replaced bydeuterium, one or more carbon atoms is replaced by ¹⁴C or one or morenitrogen atoms is replaced by ¹⁵N.

In the present specification “C₁₋₆ alkyl” refers to a straight orbranched saturated hydrocarbon chain having one to six carbon atoms.Examples include methyl, ethyl, n-propyl, isopropyl, t-butyl andn-hexyl.

The term “C₁₋₄ alkyl” has a similar meaning to the above except that itrefers to a straight or branched saturated hydrocarbon chain having oneto four carbon atoms. In the present specification, “halo” refers tofluoro, chloro, bromo or iodo.

The term “haloalkyl” refers to an alkyl group as defined abovesubstituted with one or more halo atoms. The group may have a singlehalo substituent or it may be a perhalo group. Examples includechloromethyl, trifluoromethyl, 1,2-dichloroethyl,1,1,1-tribromo-^(n)propyl and perfluoro-^(t)butyl.

The term “C₂₋₆ alkenyl” refers to a straight or branched hydrocarbonchain containing at least one carbon-carbon double bond and having twoto six carbon atoms. Examples include ethenyl, propen-1-yl andpropen-2-yl.

“C₁₋₆ alkylene” refers to a straight or branched divalent hydrocarbonlinking group having from one to six carbon atoms. Examples include—CH₂—, —CH₂CH₂—, —CH₂CH₂CH₂CH₂, —CH(CH₃)— and —CH(CH₂CH₃)—.

The terms “heterocyclic” and “heterocyclyl” refer to a non-aromaticcyclic group in which one or more ring atoms is replaced by a heteroatom selected from N, O and S. Examples include aziridine, azetidine,pyrrolidine, pyrroline, imidazoline, piperidine, piperazine, morpholine,oxetane, tetrahydrofuran and oxazoline. Further examples include fusedor bridged ring systems such as octahydroquinoline or nor-tropane.

The terms “heteroaromatic” and “heteroaryl” in the context of thespecification refers to a ring system with aromatic character havingfrom 5 to 10 ring atoms (unless otherwise specified), at least one ofwhich is a heteroatom selected from N, O and S, and containing one ringor two fused rings. Where a heteroaryl group contains more than onering, not all rings must be fully aromatic in character. Examples ofmonocyclic heteroaryl groups include pyridine, pyrimidine, furan,thiophene, pyrrole, pyrazole, imidazole, oxazole, isoxazole, thiazoleand isothiazole. Examples of bicyclic fully aromatic heteroaryl groupsinclude quinoline, isoquinoline, indole, benzofuran, benzimidazole andbenzothiazole. Examples of bicyclic heteroaryl groups in which one ringis not fully aromatic in character include dihydroquinolines,tetrahydroquinoline, tetrahydroisoquinoline, chromene, chromane,benzimidazoline, benzomorpholine, isoindoline and indoline.

Salts of the compounds of the present invention include salts of organicacids, especially carboxylic acids, including but not limited toacetate, trifluoroacetate, lactate, gluconate, citrate, tartrate,maleate, malate, pantothenate, adipate, alginate, aspartate, benzoate,butyrate, digluconate, cyclopentanate, glucoheptanate, glycerophosphate,oxalate, heptanoate, hexanoate, fumarate, nicotinate, pamoate,pectinate, 3-phenylpropionate, picrate, pivalate, proprionate, tartrate,lactobionate, pivolate, camphorate, undecanoate and succinate, organicsulfonic acids such as methanesulfonate, ethanesulfonate,2-hydroxyethane sulfonate, camphorsulfonate, 2-naphthalenesulfonate,benzenesulfonate, p-chlorobenzenesulfonate and p-toluenesulfonate; andinorganic acids such as hydrochloride, hydrobromide, hydroiodide,sulfate, bisulfate, hemisulfate, thiocyanate, persulfate, phosphoric andsulfonic acids.

Where appropriate, pharmaceutically or veterinarily acceptable salts ofthe compounds of general formula (I) or (Ia) may also include basicaddition salts salts such as sodium, potassium, calcium, aluminium,zinc, magnesium and other metal salts as well as choline,diethanolamine, ethanolamine, ethyl diamine and other well-known basicaddition salts.

Salts will preferably be pharmaceutically or veterinarily acceptable butother salts may still be valuable as intermediates.

In suitable compounds of the present invention, each R¹ is independentlyhydrogen, halo, nitro, C₁₋₄ alkyl, C₁₋₄ haloalkyl or OR⁵. R⁵ is asdefined above but is more suitably hydrogen, methyl or ethyl, either ofwhich may optionally be substituted by one or more halo substituents.

In more suitable compounds of general formula (I), each R¹ isindependently hydrogen, halo, nitro, methyl, trifluoromethyl, OH,methoxy or trifluoromethoxy.

Suitably m is 1, 2 or 3.

In suitable compounds of general formula (I), A is phenyl.

In some suitable compounds of the present invention, R² is hydrogen,methyl or ethyl. Compounds in which R² is hydrogen are particularlysuitable.

The linker group Q is suitably a straight or branched C₁₋₆ alkylenegroup.

More suitably, Q is a group (CH₂)_(p), where p is an integer of 1 to 4.In still more suitable compounds, p is 2 or 3.

In some suitable compounds of general formula (I), R³ is phenyl or a 5-or 6-membered heteroaryl group either of which may optionally besubstituted with one or more substituents selected from C₁₋₄ alkyl, C₁₋₄haloalkyl, or OH.

More suitably, in this embodiment, R³ is phenyl or a 5- or 6-memberednitrogen containing heteroaryl group such as pyridyl, pyrrolyl,pyrimidinyl, imdizolyl or triazolyl, any of which may optionally besubstituted as set out above.

Still more suitably, R³ is phenyl or pyridyl, especially pyridyl.

In other suitable compounds, R³ is NR⁷R⁸, wherein each of R⁷ and R⁸ isindependently hydrogen or C₁₋₆ alkyl or R⁷ and R⁸ together with thenitrogen atom to which they are attached may form a 5 to 7 memberedheterocyclic ring optionally containing one or more other heteroatomsselected from N, O and S.

In more suitable compounds of this embodiment R⁷ and R⁸ together withthe nitrogen atom to which they are attached may form a 5 to 7 memberedheterocyclic ring optionally containing one or more other heteroatomsselected from N, O and S.

In particularly suitable compounds, R³ is a group NR⁷R⁸, which is a 5-or 6-membered nitrogen containing heterocyclic ring. Examples ofparticularly suitable R³ groups of this type include morpholine,piperidine, piperazine, pyrrolidine, pyrazolidine and imidazoline, withmorpholine, piperidine, piperazine and pyrrolidine being especiallysuitable.

In some suitable compounds of general formula (I), each R⁴ isindependently hydrogen, halo, nitro, cyano, C₁₋₄ alkyl, C₁₋₄ haloalkylor OR⁹; where R⁹ is hydrogen, C₁₋₄ alkyl or C₁₋₄ haloalkyl.

Still more suitably, each R⁴ is independently hydrogen, halo or methylor ethyl, either of which is optionally substituted by one or more halosubstituents.

Suitably, n is 1, 2 or 3.

In suitable compounds of formula (Ia), each R¹ is independently halo,nitro, C₁₋₄ alkyl, C₁₋₄ haloalkyl or OR⁵. R⁵ is as defined above informula (Ia) but is suitably hydrogen, or methyl or ethyl, either ofwhich may optionally be substituted by one or more halo substituents.

In more suitable compounds of general formula (Ia), each R¹ isindependently halo, nitro, methyl, trifluoromethyl, OH, methoxy ortrifluoromethoxy. In still more suitable compounds of general formula(Ia), at least one R¹ is fluoro, and more suitably, 2-fluoro. In othermore suitable compounds of general formula (Ia), at least one R¹ is OR⁵.Suitably, R⁵ is C₁₋₆ alkyl, and more suitably, methyl.

Suitably, in compounds of general formula (Ia), m is 1 or 2.

In some suitable compounds of general formula (Ia), R³ is morpholinyl,piperidinyl, piperazinyl and pyrrolidinyl, with morpholinyl beingespecially suitable.

In some suitable compounds of general formula (Ia), p is 2 or 3.

In some suitable compounds of general formula (Ia), each R⁴ isindependently C₁₋₄ alkyl or OR⁹. R⁹ is as defined above in formula (Ia)but is suitably hydrogen, C₁₋₄ alkyl or C₁₋₄ haloalkyl. More suitably,each R⁴ is independently methyl or methoxy.

Suitably, in compounds of general formula (Ia), n is 0 or 1, and moresuitably, 0.

Particularly suitable compounds of general formula (I) or (Ia) include:

-   2-[(3-fluorobenzoyl)amino]-N-(2-morpholin-4-ylethyl)benzamide;-   2-[(4-fluorobenzoyl)amino]-N-(2-morpholin-4-ylethyl)benzamide;-   2-[(2-methoxybenzoyl)amino]-N-(2-morpholin-4-ylethyl)benzamide;-   2-[(3-methoxybenzoyl)amino]-N-(2-morpholin-4-ylethyl)benzamide,-   2-[(4-methoxybenzoyl)amino]-N-(2-morpholin-4-ylethyl)benzamide;-   2-[(2-nitrobenzoyl)amino]-N-(2-morpholin-4-ylethyl)benzamide;-   2-[(3-nitrobenzoyl)amino]-N-(2-morpholin-4-ylethyl)benzamide;-   2-[(4-nitrobenzoyl)amino]-N-(2-morpholin-4-ylethyl)benzamide;-   2-[(2-methylbenzoyl)amino]-N-(2-morpholin-4-ylethyl)benzamide;-   2-benzamido-N-(2-morpholin-4-ylethyl)benzamide;-   3,4-dimethoxy-N-(2-[(2-morpholin-4-ylethyl)carbamoyl]phenyl)benzamide;-   3,5-dimethoxy-N-(2-[(2-morpholin-4-ylethyl)carbamoyl]phenyl)    benzamide;-   3,4,5-trimethoxy-N-(2-[(2-morpholin-4-ylethyl)carbamoyl]phenyl)    benzamide;-   3,5-difluoro-N-(2-[(2-morpholin-4-ylethyl)carbamoyl]phenyl)benzamide;-   2,6-difluoro-N-(2-[(2-morpholin-4-ylethyl)    carbamoyl]phenyl)benzamide;-   2,4-difluoro-N-(2-[(2-morpholin-4-ylethyl)carbamoyl]phenyl)benzamide;-   2-[(2-fluorobenzoyl)amino]-N-(2-morpholin-4-ylpropyl)benzamide;-   2-[(4-fluorobenzoyl)amino]-N-(pyridin-3-ylmethyl)benzamide;-   2-[(4-fluorobenzoyl)amino]-N-(pyrrolidin-3-ylmethyl)benzamide;-   2-[(4-fluorobenzoyl)amino]-N-(piperidin-3-ylmethyl)benzamide;-   2-[(4-fluorobenzoyl)amino]-N-(piperazin-3-ylmethyl)benzamide;-   N-(2-aminoethyl)-2-(2-fluorobenzamido) benzamide;-   2-[(2-fluorobenzoyl)amino]-3-methyl-N-(2-morpholin-4-ylethyl)benzamide;-   2-[(2-fluorobenzoyl)amino]-3-methoxy-N-(2-morpholin-4-ylethyl)benzamide;-   2-[(2-fluorobenzoyl)amino]-5-iodo-N-(2-morpholin-4-ylethyl)benzamide;-   2-[(2-fluorobenzoyl)amino]-2-chloro-N-(2-morpholin-4-ylethyl)benzamide;-   2-fluoro-N-(2-((2-morpholinoethyl)carbamoyl)phenyl)benzamide;    and their pharmaceutically or veterinarily acceptable salts.

Compounds of general formula (I) may be prepared by any suitable route.For example, compounds of general formula (I) may be prepared fromcompound of general formula (II):

wherein A, R¹, m, R⁴ and n are as defined for general formula (I);by reaction with a compound of general formula (III):

wherein Q, R² and R³ are as defined for general formula (I).

Similarly, compounds of general formula (Ia) may be prepared by anysuitable route. For example, compounds of general formula (Ia) may beprepared from compound of general formula (IIa):

by reaction with a compound of general formula (IIIa):

wherein R¹, m, R³, R⁴ and n are as defined for general formula (Ia).

These processes form a further aspect of the invention.

These reactions are carried out in the presence of a base, typically anon-nucleophlic base, for example a tertiary amine such asN,N-diisopropylethylamine. These reactions may be conducted in anorganic solvent such as N,N-dimethylformamide and at a temperature of15-30° C., more typically at about 18-25° C. (room temperature).

Amines of general formula (III) or (IIIa) are well known and are readilyavailable or may be prepared by literature methods well known to thoseof skill in the art.

Compounds of general formula (II) may be prepared from compounds ofgeneral formula (IV):

wherein R⁴ and n are as defined for general formula (I);by reaction with compounds of general formula (V):

wherein A, R¹ and m are as defined for general formula (I) and X is aleaving group, typically a halo group such as chloro.

Similarly, compounds of general formula (Ha) may be prepared fromcompounds of general formula (IVa):

by reaction with compounds of general formula (Va):

wherein R¹, m, R⁴ and n are as defined for general formula (Ia) and X isa leaving group, typically a halo group such as chloro.

These reactions may be carried out in an organic solvent such aspyridine and at a temperature of 15-30° C., more typically at about18-25° C. (room temperature).

Compounds of general formulae (IV), (IVa), (V) and (Va) are well knownand are readily available or may be prepared by literature methods wellknown to those of skill in the art.

As discussed above, the compounds of the present invention are Bcl3inhibitors and are therefore of use in the treatment of cancer, forexample leukaemias and lymphomas, such as anaplastic large celllymphomas (ALCLs), classic Hodgkin lymphomas (cHL) and non-Hodgkin'slymphoma; and solid tumour cancers, such as breast cancer, melanoma,lung cancer, pancreatic cancer, oesophageal cancer, colorectal cancer,nasopharyngeal carcinoma, ovarian, prostate and hepatocarcinomas.

Therefore, in a further aspect of the invention there is provided acompound of general formula (I), (Ia) or2-[(2-fluorobenzoyl)amino-N-2-morpholin-4-ylethyl)benzamide for use inmedicine. In particular, there is provided a compound of general formula(I) or (Ia) for use in the treatment of cancer, for example leukaemiasand lymphomas, such as anaplastic large cell lymphomas (ALCLs), classicHodgkin lymphomas (cHL) and non-Hodgkin's lymphoma; and solid tumourcancers, such as breast cancer, melanoma, lung cancer, pancreaticcancer, oesophageal cancer, colorectal cancer, ovarian cancer, prostatecancer, nasopharyngeal carcinoma, and hepatocarcinomas.

In a further aspect of the invention, there is provided the use of acompound of general formula (I), (Ia) or2-[(2-fluorobenzoyl)amino-N-2-morpholin-4-ylethyl)benzamide in thepreparation of an agent for the treatment of cancer, for exampleleukaemias and lymphomas, such as anaplastic large cell lymphomas(ALCLs), classic Hodgkin lymphomas (cHL) and non-Hodgkin's lymphoma; andsolid tumour cancers, such as breast cancer, melanoma, lung cancer,pancreatic cancer, oesophageal cancer, colorectal cancer, ovariancancer, prostate cancer, nasopharyngeal carcinoma and hepatocarcinomas.

The compounds may be used either in human or in veterinary medicine andthe patient may be any mammal but especially a human.

The invention also provides a method for the treatment of cancer, forexample leukaemias and lymphomas, such as anaplastic large celllymphomas (ALCLs), classic Hodgkin lymphomas (cHL) and non-Hodgkin'slymphoma; and solid tumour cancers, such as breast cancer, melanoma,lung cancer, pancreatic cancer, oesophageal cancer, colorectal cancer,ovarian cancer, prostate cancer, nasopharyngeal carcinoma, andhepatocarcinomas, the method comprising administering to a patient inneed of such treatment an effective amount of a compound of generalformula (I), (Ia) or2-[(2-fluorobenzoyl)amino-N-2-morpholin-4-ylethyl)benzamide.

The compounds are particularly useful for the treatment or prevention ofmetastasis in cancers.

Suitably, the cancer is breast cancer, more particularly triple negativebreast cancer or HER2 enriched breast cancer. The compounds of formula(I) or (Ia) have been shown to be particularly effective in preventingor treating metastasis in models of these breast cancer subtypes.However this does not preclude its relevance or efficacy in metastaticdisease in other tumour types. Moreover, our experimental evidence inhuman cancer cell lines indicates that there may also be beneficialtherapeutic effects of Bcl-3 suppression on tumour cell viability, asboth genetic suppression of Bcl-3 and use of compounds of formula (I) or(Ia) partially but significantly reduce tumour cell numbers in vitro.

The compounds of the invention will generally be formulated foradministration by a desired route.

Therefore, in a further aspect of the invention there is provided apharmaceutical composition comprising a compound of general formula (I),(Ia) or 2-[(2-fluorobenzoyl)amino-N-2-morpholin-4-ylethyl)benzamidetogether with a pharmaceutically or veterinary acceptable excipient orcarrier.

The carrier, or, if more than one be present, each of the carriers, mustbe acceptable in the sense of being compatible with the otheringredients of the formulation and not deleterious to the recipient.

The formulations include those suitable for oral, rectal, nasal, topical(including eye drops, buccal and sublingual), vaginal or parenteral(including subcutaneous, intramuscular, intravenous and intradermal)administration and may be prepared by any methods well known in the artof pharmacy.

The route of administration will depend upon the condition to be treatedbut preferred compositions are formulated for parenteral administration.

Parenteral formulations will generally be sterile.

The composition may be prepared by bringing into association the abovedefined active agent with the carrier. In general, the formulations areprepared by uniformly and intimately bringing into association theactive agent with liquid carriers or finely divided solid carriers orboth, and then if necessary shaping the product. The invention extendsto methods for preparing a pharmaceutical composition comprisingbringing a compound of general formula (I), (Ia) or2-[(2-fluorobenzoyl)amino-N-2-morpholin-4-ylethyl)benzamide intoassociation with a pharmaceutically or veterinarily acceptable excipientor carrier.

Typically, the dose of the compound will be about 0.01 to 100 mg/kg; soas to maintain the concentration of drug in the plasma at aconcentration effective to inhibit Bcl3. The precise amount of acompound of general formula (I), (Ia) or2-[(2-fluorobenzoyl)amino-N-2-morpholin-4-ylethyl)benzamide which istherapeutically effective, and the route by which such compound is bestadministered, is readily determined by one of ordinary skill in the artby comparing the blood level of the agent to the concentration requiredto have a therapeutic effect.

The compound of general formula (I), (Ia) or2-[(2-fluorobenzoyl)amino-N-2-morpholin-4-ylethyl)benzamide may be usedin combination with one or more additional active agents which areuseful in the treatment of cancer.

Without limitation, examples of such agents include first-line oradjuvant anti-hormone, radio- or chemo-therapeutics aimed at targetingthe primary lesion or suppressing late stage disease progression; forexample, anti-HER2 agents such as trastuzumab and pertuzumab andstandard adjuvant therapy regimens such as 5-fluorouracil, doxorubicin,and cyclophosphamide (FAC); 5-fluorouracil, epirubicin, andcyclophosphamide (FEC); and doxorubicin and cyclophosphamide (AC);cyclophosphamide, methotrexate, and 5-fluorouracil (CMF); and docetaxel,doxorubicin, cyclophosphamide (TAC). Other suitable agents for use incombination with the compounds of the invention areanti-angiogenic/antimetastatic agents such as bevacizumab (Avastin).

Other features of the present invention will become apparent from thefollowing examples. Generally speaking, the invention extends to anynovel one, or any novel combination, of the features disclosed in thisspecification (including the accompanying claims and drawings). Thus,features, integers, characteristics, compounds or chemical moietiesdescribed in conjunction with a particular aspect, embodiment or exampleof the invention are to be understood to be applicable to any otheraspect, embodiment or example described herein, unless incompatibletherewith.

Moreover, unless stated otherwise, any feature disclosed herein may bereplaced by an alternative feature serving the same or a similarpurpose.

The Invention will now be described by way of example only withreference to the Examples below and to the following Figures wherein:

FIG. 1. Cell toxicity of Compound 1a. MCF-10A [A], MDA-MB-231 [B] andSKBR3 [C] breast cancer cells were cultivated with Compound 1a over arange of molarities in adherent growth conditions. Cell viability wasdetermined after 24 hrs by the Cell Titre Blue viability assay andresulting fluorescence was normalised against fluorescence of controlcells treated with DMSO in relevant concentration. Data representaverage of six wells and error bars represent ±SEM. Dose response curveswere generated using GraphPad software. The IC₅₀ for each cell line isshown to the left of each graph.

FIG. 2. Establishing the ability of Compound 1a to inhibit Bcl3 bindingto its cognate protein partner NFKB1 (p50) by Indirect Sandwich ELISAassay. HEK-293 cells over-expressing FLAG-tagged Bcl-3 were cultivatedwith compound 1a or DMSO in a range of molarities in normal adherentgrowth conditions for 24 hrs. Cell lysates were prepared undernon-denaturing conditions. [A] Indirect sandwich ELISA assay wasperformed on anti-FLAG coated ELISA plates using p50 antibody.Absorbance was measured at 405 nm and normalised to that of DMSOcontrol. Error bars represent ±SEM of three independent wells. The doseresponse curve was generated using GraphPad software. The IC₅₀ forCompound 1a is shown inset. [B] and [C] Indirect ELISA assay wasperformed on FLAG coated ELISA plates using Bcl-3 antibody. Absorbancewas measured at 405 nm. Error bars represent ±SEM of three independentwells.

FIG. 3. Establishing the ability of Compound 1a to inhibit NF-κBsignalling by NF-κB promoter-reporter (luciferase) assay. MDA-MB-231overexpressing Bcl-3 [A], HEK-293 cells overexpressing Bcl-3 [B] andHEK-293 overexpressing Bcl-3 and p52 [C] were cultivated with Compound1a or DMSO control in a range of molarities for 24 hrs before beingtransfected with NF-κB luciferase reporter for 48 hrs together withcontrols. NF-κB activity is represented as a % of DMSO control. Errorbars represent ±SEM of three independent experiments. The dose responsecurve was generated using GraphPad software. IC₅₀s for each cell lineare shown inset.

FIG. 4. Establishing the effect of Compound 1a on cell motility byBoyden Chamber assay. [A] MDA-MB-231 cells overexpressing Bcl-3 werecultivated with compound 1a (10 μM, 1 μM, 100 nM, 10 nM) or DMSO incorresponding concentration in normal adherent growth conditions for 24hrs before being seeded onto Boyden motility chambers for 24 hrs inparallel with cells overexpressing the Bcl-3 binding mutant ANK M123.Migrated cells were counted from three fields of view of each of threereplicate Boyden chambers. Error bars represent ±SEM. The dose responsecurve was generated using GraphPad software. The IC₅₀ is shown inset.[B] Representative images of migrated cells for MDA-MB-231 cellsoverexpressing Bcl-3 and treated with either compound 1a or DMSO. Scalebars represent 200 μm.

FIG. 5. Cell toxicity of series 3 compounds. MDA-MB-231 cells [A to C]and HEK-293 cells [D to F] were cultivated with compounds from series 1,2 or 3 (10 nM, 100 nM, 1 μM and 10 μM) in adherent growth conditions.Cell viability was determined after 24 hrs by the Cell Titre Blueviability assay and resulting fluorescence was normalised against thatof respective cells treated with DMSO under the same conditions. Datarepresent average of six wells and error bars represent ±SEM.

FIG. 6. NF-κB assay in MDA-MB-231 cells with series 1 to 3 compounds.MDA-MB-231 cells overexpressing Bcl-3 were cultivated with compoundsfrom series 1 [A], 2 [B] and 3 [C] at 1 μM concentration or DMSO controlfor 24 hrs before being transfected with NF-κB luciferase reporter for48 hrs together with controls. NF-κB activity is plotted on a log scaleas relative light units and normalised to the NF-κB activity ofMDA-MB-231 cells not overexpressing Bcl-3. Error bars represent ±SEM ofthree independent transfections. (T-test, *=p<0.05 as compared toMDA-MB-231 Bcl-3 WT).

FIG. 7. Establishing the effect of selected analogues on NF-κBsignalling by luciferase-reporter assay in MDA-MB-231 cells. MDA-MB-231cells overexpressing Bcl-3 were cultivated with analogues 1f [A], 2a[B], 2c [C] and 3a [D] or DMSO control in a range of molarities for 24hrs before being transfected with NF-κB luciferase reporter for 48 hrstogether with controls. NF-κB activity is represented as a % of DMSOcontrol. Error bars represent ±SEM of three independent transfections.The dose response curve was generated using GraphPad software. The IC₅₀for each of the analogues are shown on the left of each graph.

FIG. 8. Establishing the effect of selected analogues on Bcl-3 bindingto p50 by Indirect Sandwich ELISA Assay. HEK-293 cells overexpressingFLAG-tagged Bcl-3 were cultivated with compound 1f [A], 2a [B], 2c [C]and 3a [D] or DMSO in a range of molarities in normal adherent growthconditions for 24 hrs. Cell lysates were prepared under non-denaturingconditions. Indirect sandwich ELISA assay was performed on FLAG coatedELISA plates using p50 antibody. Absorbance was measured at 405 nm andnormalised to that of DMSO control. Error bars represent ±SEM of threeindependent wells. The dose response curve was generated using GraphPadsoftware. IC₅₀s are shown for each analogue to the left of each graph.[E-H]. Indirect ELISA assay was performed on FLAG coated ELISA platesusing Bcl-3 antibody. Absorbance was measured at 405 nm. Error barsrepresent ±SEM of three independent wells.

FIG. 9. Establishing IC₅₀ the effect of selected analogues on cellmotility by Boyden chamber assay. MDA-MB-231 cells overexpressing Bcl-3were cultivated with compounds 1f [A], 2a [B], 2c [C] and 3a [D] or DMSOin a range of molarities in normal adherent growth conditions for 24 hrsbefore being seeded onto Boyden motility chambers for 24 hrs in parallelwith MDA-MB-231 cells expressing the Bcl-3 binding mutant ANK M123.Migrated cells were counted from three fields of view of each of threereplicate Boyden chambers. Error bars represent ±SEM. The dose responsecurve was generated using GraphPad software. The IC₅₀s for each analogueare shown to the left of each graph.

FIG. 10. Establishing the effect of analogue if on cell viability.MDA-MB-231 cells were cultivated with the analogue if over a range ofmolarities in adherent growth conditions. Cell viability was determinedafter 24 hrs by the Cell Titre Blue viability assay and resultingfluorescence was normalised against fluorescence of control cellstreated with DMSO in relevant concentration. Data represent average ofsix wells and error bars represent ±SEM. Dose response curves weregenerated using GraphPad software. The IC₅₀ is shown to the left of thegraph.

FIG. 11. Biological evaluation of di- and tri-substituted analogues [A]and [B]. MDA-MB-231 cells were cultivated with di- and tri-substitutedcompounds from series 1 (1l-1q) over a range of molarities in adherentgrowth conditions. Cell viability was determined after 24 hrs by theCell Titre Blue viability assay and resulting fluorescence wasnormalised against that of respective cells treated with DMSO under thesame conditions. Data represent average of six wells and error barsrepresent ±SEM. [C] MDA-MB-231 cells overexpressing Bcl-3 werecultivated with compounds from series 1 (1l-1q) at 1 μM concentration orDMSO control for 24 hrs before being transfected with NF-κB luciferasereporter for 48 hrs together with controls. NF-κB activity is plotted ona log scale as relative light units and normalised to the NF-κB activityof MDA-MB-231 cells. Error bars represent ±SEM of three independenttransfections. (T-test, *=p<0.05 as compared to MDA-MB-231).

FIG. 12. Establishing the effect of selected di- and tri-substitutedanalogues on NF-κB activity by luciferase-reporter assay in MDA-MB-231cells. MDA-MB-231 cells overexpressing Bcl-3 were cultivated withanalogues 10 [A], 1p [B], 1q [c] or DMSO control at a range ofmolarities for 24 hrs before being transfected with NF-κB luciferasereporter for 48 hrs together with controls. NF-κB activity isrepresented as a % of DMSO control. Error bars represent ±SEM of threeindependent transfections. The dose response curve was generated usingGraphPad software. IC₅₀s are shown to the left of each graph.

EXAMPLES

Synthesis of Compounds

The compounds of the invention were synthesised according to the generalmethod shown in Scheme 1.

Scheme 1 illustrates the cyclocondensation reaction of the anthranilicacid derivative (IV) and compound (V) which gives rise to theIntermediate (II). In the second step the intermediate (II) was reactedwith the amine (III) to give the final product (I).

All chemicals used in this investigation were obtained from commercialsuppliers (Sigma Aldrich) and were used without further purification.All glassware were washed and dried before each experiment. Solventswere evaporated using the Buchi Rotavapor. Melting points were measuredon a Griffin apparatus using a capillary method.

The ¹H, ¹³C NMR spectra were recorded on a Bruker AVANCE 500spectrometer at 500 and 126 MHz respectively, at 25° C. Chemical shifts(6) are reported in parts per million (ppm). J values are reported inHertz (Hz). Dimethyl sulfoxide (DMSO) was used as a solvent. Usedabbreviations include s (singlet), d (doublet), t (triplet), q(quadruplet), m (multiplet), dd (doublet of doublets), dt (doublet oftriplets), td (triplet of doublets).

TLC was performed using Merck TLC silica gel 60 plates F254 (40-60 μM)with detection by UV light (254-366 nm).

Mass spectrometry was run using electron ionisation (EI) andelectrospray (ES) on a Waters GCT Premier or a Waters LCT Premier XE,respectively. The mass spectrometry was performed as a service by Schoolof Chemistry, Cardiff University. Elemental analysis (CHN) microanalysiswas performed as a service by MEDAC Ltd, Surrey. High resolution massspectrometry was performed on LTQ Orkitrap XL by the EPSRC National MassSpectrometry Service (Swansea, UK).

General Method for Step 1

In the first step, an anthranilic acid derivative (IV) was dissolved inpyridine (5 ml) and 2.2 equivalent of a substituted benzoyl chloride (V)and stirred at r.t. The reaction was monitored by TLC and stopped afterapproximately an hour after complete disappearance of the anthranilicacid (V). The reaction mixture was poured into 10% solution of sodiumcarbonate (3 g sodium carbonate, 27 ml distilled water). The formedprecipitate was collected by filtration under reduced pressure asintermediate (II).

General Method for Step 2

In the second step, to a stirred solution of intermediate (II) DMF (10ml) were added 2 equivalents of DIPEA and 2.2 equivalents of amine(III). The reaction mixture was stirred at r.t overnight. The completedisappearance of starting material (II) was monitored by TLC. Thereaction mixture was dissolved in DCM and washed three times with water.The product was evaporated under reduced pressure and the obtained solidwas recrystallized from ethanol. All synthesised compounds were analysedby ¹H, ¹³C NMR spectra and mass spectrometry. The purity of finalcompounds was also confirmed by elemental analysis.

Example 1 Synthesis of Compounds of Series 1

The compounds of series 1 have the general formula

Compound R¹ 1a 2-F 1b 3-F 1c 4-F 1d 2-OCH₃ 1e 3-OCH₃ 1f 4-OCH₃ 1g 2-NO₂1h 3-NO₂ 1i 4-NO₂ 1j 2-CH₃ 1k H 1l 3,4-OCH₃ 1m 3,5-OCH₃ 1n 3,4,5-OCH₃ 1o3,5-F 1p 2,6-F 1q 2,4-F

Step 1—Synthesis of Intermediates Synthesis of2-(2-fluorophenyl)-4H-3,1-benzoxazin-4-one (Intermediate 1a)

Chemical Formula: C14H8FNO2, Molecular Weight: 241.22

The synthetic procedure followed the general method above usinganthranilic acid (0.50 g, 3.65 mmol) dissolved in pyridine (5 ml) and2.2 equivalent of 2-fluorobenzoyl chloride (0.96 ml, 8.02 mmol).Collected as a white solid, yield 85% (0.75 g), mp 97° C.

¹H NMR (500 MHz, DMSO-d6) δ 8.18 (dd, J=8.0, 1.5 Hz, 1H, ArH), 8.10 (td,J=7.8, 1.8 Hz, 1H, ArH), 7.98 (td, J=9.1, 2.2 Hz, 1H, ArH), 7.74 (d,J=7.9 Hz, 1H, ArH), 7.68 (td, J=7.6, 1.2 Hz, 2H, ArH), 7.47-7.40 (m, 2H,ArH).

¹³C NMR (126 MHz, DMSO-d6) δ 160.48 (d, J_(C-F)=258.3 Hz, ArC—F), 158.66(ArC═O), 154.23 (ArC), 146.03 (ArC), 136.88 (ArCH), 134.52 (d,J_(C-F)=8.8 Hz, ArCH), 131.10 (ArCH), 129.02 (ArCH), 127.98 (ArCH),127.03 (ArCH), 124.83 (d, J_(C-F)=3.8 Hz, ArCH), 118.66 (d, J_(C-F)=10.1Hz, ArC), 117.21 (d, J_(C-F)=21.4 Hz, ArCH), 116.92 (ArC).

MS (APCI⁺): 242.05 [M+1].

Synthesis of 2-(3-fluorophenyl-4H-3,1-benzoxazin-4-one (Intermediate 1b)

Chemical Formula: C14H8FNO2, Molecular Weight: 241.22

The synthetic procedure followed the general method above usinganthranilic acid (0.50 g, 3.65 mmol) dissolved in pyridine (5 ml) and2.2 equivalent of 3-fluorobenzoyl chloride (0.98 ml, 8.02 mmol).Collected as a white solid, yield 80% (0.71 g), mp 96° C.

¹H NMR (500 MHz, DMSO-d6) δ 8.48 (d, J=8.3 Hz, 1H, ArH), 8.17 (d, J=7.9Hz, 1H, ArH), 8.06-7.93 (m, 2H, ArH), 7.87 (d, J=10.4 Hz, 1H, ArH), 7.81(d, J=10.1 Hz, 1H, ArH), 7.77-7.48 (m, 1H, ArH), 7.27 (t, J=7.7 Hz, 1H,ArH).

¹³C NMR (126 MHz, DMSO-d6) δ 162.17 (d, J_(C-F)=245.7 Hz, ArC—F), 158.56(ArC═O), 145.93 (ArC), 139.71 (ArC), 136.89 (ArCH), 134.20 (ArCH),131.28 (d, J J_(C-F)=8.2 Hz, ArCH), 130.64 (ArCH), 128.90 (ArCH), 127.00(ArCH), 123.69 (d, J_(C-F)=2.8 Hz, ArCH), 121.14 (ArC), 119.35 (d,J_(C-F)=88.2 Hz, ArCH), 117.37 (d, J_(C-F)=85.7 Hz, ArC).

MS (APCI⁺): 242.06 [M+1]. O N O F

Synthesis of 2-(4-fluorophenyl)-4H-3,1-benzoxazin-4-one (Intermediate1c)

Chemical Formula: C14H8FNO2 Molecular Weight: 241.22

The synthetic procedure followed the general method above usinganthranilic acid (0.50 g, 3.65 mmol) dissolved in pyridine (5 ml) and2.2 equivalent of 4-fluorobenzoyl chloride (0.95 ml, 8.02 mmol).Collected as a white solid, yield 91% (0.80 g), mp 159° C.

¹H NMR (500 MHz, DMSO-d6) δ 8.26 (td, J=14.1, 5.4 Hz, 2H, ArH), 8.16(dd, J=8.0, 1.5 Hz, 1H, ArH), 7.96 (t, J=15.8 Hz, 1H, ArH), 7.72 (d,J=8.1 Hz, 1H, ArH), 7.63 (td, J=7.6, 1.2 Hz, 1H, ArH), 7.48-7.40 (m, 2H,ArH).

¹³C NMR (126 MHz, DMSO-d6) δ 164.74 (d, J_(C-F)=251.4 Hz, ArC—F), 158.72(ArC═O), 155.58 (ArC), 146.18 (ArC), 136.87 (ArCH), 130.53 (d,J_(C-F)=9.4 Hz, ArCH), 128.57 (ArCH), 128.05 (ArCH), 126.83 (d,J_(C-F)=3.8 Hz, ArCH), 126.61 (ArC), 116.81 (ArC), 116.17 (d,J_(C-F)=22.3 Hz, ArCH).

MS (APCI⁺): 242.06 [M+1].

Synthesis of 2-(2-methoxyphenyl)-4H-3,1-benzoxazin-4-one (Intermediate1d)

Chemical Formula: C15H11NO3 Molecular Weight: 253.25

The synthetic procedure followed the general method above usinganthranilic acid (0.50 g, 3.65 mmol) dissolved in dry pyridine (5 ml)under anhydrous conditions in nitrogen atmosphere and 2.2 equivalent of2-methoxybenzoyl chloride (1.19 ml, 8.02 mmol). Collected as a whitesolid, yield 98% (0.91 g), mp 107° C.

¹H NMR (500 MHz, DMSO-d6) δ 8.17 (dd, J=7.9, 1.6 Hz, 1H, ArH), 7.97 (td,16.0, 1.1 Hz, 1H, ArH), 7.78 (dd, J=7.7, 1.8 Hz, 1H, ArH), 7.71 (d, 7.9Hz, 1H, ArH), 7.66 (td, J=7.6, 1.2 Hz, 1H, ArH), 7.60 (td, J=8.8, 1.8Hz, 1H, ArH), 7.24 (d, J=8.4 Hz, 1H, ArH), 7.12 (t, J=7.5 Hz, 1H, ArH),3.88 (s, 3H, OCH3).

¹³C NMR (126 MHz, DMSO-d6) δ 159.15 (ArC═O), 157.89 (ArC), 157.16 (ArC),146.31 (ArC), 136.88 (ArCH), 133.24 (ArCH), 130.97 (ArCH), 128.81(ArCH), 127.90 (ArCH), 126.89 (ArCH), 120.36 (ArCH), 120.27 (ArC),116.53 (ArC), 112.56 (ArCH), 55.66 (OCH3).

MS (APCI⁺): 254.07 [M+1].

Synthesis of 2-(3-methoxyphenyl)-4H-3,1-benzoxazin-4-one (Intermediate1e)

Chemical Formula: C15H11NO3 Molecular Weight: 253.25 The syntheticprocedure followed the general method above using anthranilic acid (0.50g, 3.65 mmol) dissolved in dry pyridine (5 ml) under anhydrousconditions in nitrogen atmosphere and 2.2 equivalent of 3-methoxybenzoylchloride (1.13 ml, 8.02 mmol). Collected as a white solid, yield 97%(0.90 g), mp 103° C.

¹H NMR (500 MHz, DMSO-d6) δ 8.14 (dd, J=7.9, 1.5 Hz, 1H, ArH), 7.94 (t,J=7.9 Hz, 1H, ArH), 7.76 (d, J=7.7, Hz, 1H, ArH), 7.71 (d, J=8.1 Hz, 1H,ArH), 7.66-7.58 (m, 2H, ArH), 7.50 (t, J=8.0 Hz, 1H, ArH), 7.21 (dd,J=8.4, 2.6 Hz, 1H, ArH), 3.85 (s, 3H, OCH3).

¹³C NMR (126 MHz, DMSO-d6) δ 159.42 (ArC═O), 158.79 (ArC), 156.10 (ArC),146.09 (ArC), 136.86 (ArCH), 131.27 (ArC), 130.16 (ArCH), 128.64 (ArCH),128.01 (ArCH), 126.90 (ArCH), 120.15 (ArCH), 118.72 (ArCH), 116.79(ArC), 112.36 (ArCH), 55.35 (OCH3).

MS (APCI⁺): 254.07 [M+1].

Synthesis of 2-(4-methoxyphenyl)-4H-3,1-benzoxazin-4-one (Intermediate1f)

Chemical Formula: C15H11NO3 Molecular Weight: 253.25

The synthetic procedure followed the general method above usinganthranilic acid (0.50 g, 3.65 mmol) dissolved in dry pyridine (5 ml)under anhydrous conditions in nitrogen atmosphere and 2.2 equivalent of4-methoxybenzoyl chloride (1.09 ml, 8.02 mmol). Collected as a whitesolid, yield 71% (0.66 g), mp 121° C.

¹H NMR (500 MHz, DMSO-d6) δ 8.14 (td, J=17.8, 1.8 Hz, 3H, ArH), 7.93(td, J=8.5, 1.6 Hz, 1H, ArH), 7.68 (d, J=7.9 Hz, 1H, ArH), 7.59 (td,J=7.4, 1.2 Hz, 1H, ArH), 7.14 (d, J=9.0 Hz, 2H, ArH), 3.88 (s, 3H,OCH3).

¹³C NMR (126 MHz, DMSO-d6) δ 162.88 (ArC═O), 158.95 (ArC), 156.39 (ArC),146.59 (ArC), 136.80 (ArCH), 132.66 (ArCH), 129.80 (ArCH), 126.61(ArCH), 122.10 (ArCH), 120.18 (ArC), 116.55 (ArC), 114.45 (ArCH), 55.56(OCH3).

MS (APCI⁺): 254.08 [M+1].

Synthesis of 2-(2-nitrophenyl)-4H-3,1-benzoxazin-4-one (Intermediate 1q)

Chemical Formula: C14H8N2O4 Molecular Weight: 268.22

The synthetic procedure followed the general method above usinganthranilic acid (0.50 g, 3.65 mmol) dissolved in pyridine (5 ml) and2.2 equivalent of 2-nitrobenzoyl chloride (1.06 ml, 8.02 mmol).Collected as a yellow solid, yield 77% (0.75 g), mp 169° C.

¹H NMR (500 MHz, DMSO-d6) δ 8.22 (dd, J=7.8, 1.5 Hz, 1H, ArH), 8.17 (dd,J=8.0, 1.3 Hz, 1H, ArH), 8.10 (dd, J=7.6, 1.5 Hz, 1H, ArH), 8.02 (td,J=7.7, 1.6 Hz, 1H, ArH), 7.96 (td, J=7.6, 1.3 Hz, 1H, ArH), 7.91 (td,J=7.8, 1.6 Hz, 1H, ArH), 7.76-7.69 (m, 2H, ArH).

¹³C NMR (126 MHz, DMSO-d6) δ 158.20 (ArC═O), 154.50 (ArC), 148.15 (ArC),145.58 (ArC), 137.26 (ArCH), 133.60 (ArCH), 132.97 (ArCH), 131.16(ArCH), 129.61 (ArCH), 128.22 (ArCH), 127.04 (ArCH), 124.98 (ArC),124.57 (ArCH), 116.68 (ArC).

MS (APCI⁺): 269.05 [M+1].

Synthesis of 2-(3-nitrophenyl)-4H-3,1-benzoxazin-4-one (Intermediate 1h)

Chemical Formula: C14H8N2O4 Molecular Weight: 268.22

The synthetic procedure followed the general method above usinganthranilic acid (0.50 g, 3.65 mmol) dissolved in pyridine (5 ml) and2.2 equivalent of 3-nitrobenzoyl chloride (1.06 ml, 8.02 mmol).Collected as a yellow solid, yield 96% (0.94 g), mp 115° C.

¹H NMR (500 MHz, DMSO-d6) δ 8.74 (t, J=2.0 Hz, 1H, ArH), 8.48 (dd,J=8.3, 1.1 Hz, 1H, ArH), 8.37 (dd, J=8.5, 1.3 Hz, 2H, ArH), 7.99 (dd,J=7.9, 1.6 Hz, 1H, ArH), 7.91 (t, J=8.0 Hz, 1H, ArH), 7.70 (td, J=8.5,1.7 Hz, 1H, ArH), 7.30 (td, J=7.6, 1.3 Hz, 1H, ArH).

¹³C NMR (126 MHz, DMSO-d6) δ 167.70 (ArC═O), 162.76 (ArC), 148.00 (ArC),139.20 (ArC), 135.84 (ArC), 133.98 (ArCH), 133.23 (ArCH), 130.69 (ArCH),126.53 (ArCH, 124.09 (ArCH), 122.02 (ArCH), 118.95 (ArC).

MS (APCI⁺): 269.06 [M+1].

Synthesis of 2-(3-nitrophenyl)-4H-3,1-benzoxazin-4-one (Intermediate 1i)

Chemical Formula: C14H8N2O4 Molecular Weight: 268.22

The synthetic procedure followed the general method above usinganthranilic acid (0.50 g, 3.65 mmol) dissolved in pyridine (5 ml) and2.2 equivalent of 4-nitrobenzoyl chloride (1.49 g, 8.02 mmol). Collectedas a yellow solid, yield 96% (0.94 g), mp 169° C.

¹H NMR (500 MHz, DMSO-d6) δ 8.19 (t, J=7.8 Hz, 2H, ArH), 8.00 (t, J=7.8Hz, 2H, ArH), 7.79 (d, J=8.1 Hz, 2H, ArH), 7.69 (t, J=7.6 Hz, 2H, ArH).

¹³C NMR (126 MHz, DMSO-d6) δ 158.43 (ArC═O), 154.74 (ArC), 149.62 (ArC),145.77 (ArC), 136.98 (ArCH), 135.81 (ArC), 129.37 (ArCH), 129.13 (ArCH),128.13 (ArCH), 127.24 (ArCH), 124.08 (ArCH), 117.20 (ArC).

MS (APCI⁺): 269.08 [M+1].

Synthesis of 2-(o-tolyl)-4H-3,1-benzoxazin-4-one (Intermediate 1i)

Chemical Formula: C15H11NO2 Molecular Weight: 237.25

The synthetic procedure followed the general method above usinganthranilic acid (0.50 g, 3.65 mmol) dissolved in pyridine (5 ml) and2.2 equivalent of 2-methyl benzoyl chloride (1.05 ml, 8.02 mmol).Collected as a yellow solid, yield 99% (0.86 g), mp 102° C.

¹H NMR (500 MHz, DMSO-d6) δ 8.17 (d, J=8.0 Hz, 1H, ArH), 7.99-7.90 (m,2H, ArH), 7.71 (d, J=8.1 Hz, 1H, ArH), 7.65 (t, J=7.6 Hz, 1H, ArH), 7.51(t, J=7.5 Hz, 1H, ArH), 7.40 (t, J=6.9 Hz, 2H, ArH), 2.66 (s, 3H, CH3).

¹³C NMR (126 MHz, DMSO-d6) δ 159.07 (ArC═O), 157.59 (ArC), 146.17 (ArC),138.32 (ArC), 136.74 (ArCH), 131.69 (ArCH), 131.48 (ArCH), 129.88 (ArC),129.79 (ArCH), 128.69 (ArCH), 127.84 (ArCH), 126.93 (ArCH), 126.07(ArCH), 116.68 (ArC), 21.31 (CH3).

MS (APCI⁺): 238.07 [M+1].

Synthesis of 2-phenyl-4H-3,1-benzoxazin-4-one (Intermediate 1k)

Chemical Formula: C14H9NO2 Molecular Weight: 223.23

The synthetic procedure followed the general method above usinganthranilic acid (0.50 g, 3.65 mmol) dissolved in pyridine (5 ml) and2.2 equivalent of benzoyl chloride (0.93 ml, 8.02 mmol). Collected as ayellow solid, yield 95% (0.77 g), mp 91° C.

¹H NMR (500 MHz, DMSO-d6) δ 8.22 (dd, J=7.3, 1.7 Hz, 1H, ArH), 8.03 (dd,J=7.9, 1.6 Hz, 1H, ArH), 7.98 (dd, 8.1, 1.6 Hz, 2H, ArH), 7.72-7.64 (m,2H, ArH), 7.62 (dd, J=8.1, 6.6 Hz, 3H, ArH).

¹³C NMR (126 MHz, DMSO-d6) δ 168.04 (ArC═O), 164.76 (ArC), 140.25 (ArC),136.87 (ArCH), 134.33 (ArCH), 132.19 (ArCH), 130.69 (ArCH), 129.00(ArCH), 127.03 (ArCH), 123.34 (ArCH), 120.75 (ArCH), 116.99 (ArC).

MS (APCI⁺): 224.08 [M+1].

Synthesis of 2-(3,4-dimethoxyphenyl)-4H-3,1-benzoxazin-4-one(Intermediate 1l)

Chemical Formula: C16H13NO4 Molecular Weight: 283.28

The synthetic procedure followed the general method above usinganthranilic acid (0.50 g, 3.65 mmol) dissolved in pyridine (5 ml) and2.2 equivalent of 3,4-dimethoxybenzoyl chloride (1.61 g, 8.02 mmol).Collected as a white solid, yield 83% (0.86 g), mp 166° C.

¹H NMR (500 MHz, DMSO-d6) δ 8.15 (dd, J=7.8, 1.6 Hz, 1H, ArH), 7.94 (td,J=8.4, 1.6 Hz, 1H, ArH), 7.83 (dd, J=8.5, 2.1 Hz, 1H, ArH) 7.71 (dd,J=6.8, 1.7 Hz, 2H, ArH), 7.60 (td, J=8.3, 1.1 Hz, 1H, ArH), 7.18 (d,J=8.6 Hz, 1H, ArH), 3.89 (d, J=4.7 Hz, 6H, OCH3).

¹³C NMR (126 MHz, DMSO-d6) δ 159.35 (ArC═O), 158.95 (ArC), 152.82 (ArC),148.79 (ArC), 146.56 (ArC), 136.84 (ArCH), 128.09 (ArCH), 126.65 (ArCH),122.07 (ArC), 121.86 (ArCH), 116.65 (ArC), 111.56 (ArCH), 110.19 (ArCH),55.77 (OCH3), 55.63 (OCH3).

MS (EI⁺): 283.1.

Synthesis of 2-(3,5-dimethoxyphenyl)-4H-3,1-benzoxazin-4-one(Intermediate 1m)

Chemical Formula: C16H13NO4 Molecular Weight: 283.28

The synthetic procedure followed the general method above usinganthranilic acid (0.50 g, 3.65 mmol) dissolved in pyridine (5 ml) and2.2 equivalent of 3,5-dimethoxybenzoyl chloride (1.61 g, 8.02 mmol).Collected as a white solid, yield 93% (0.97 g), mp 165° C.

¹H NMR (500 MHz, DMSO-d6) δ 8.71 (dd, J=7.8, 1.6 Hz, 1H, ArH), 7.97 (t,J=7.6 Hz, 1H, ArH), 7.75 (dd, J=8.0, 2.1 Hz, 1H, ArH), 7.65 (t, J=7.6Hz, 1H, ArH), 7.31 (d, J=2.5 Hz, 2H, ArH), 6.81 (d, J=2.3 Hz, 1H, ArH),3.86 (s, 6H, OCH₃)

¹³C NMR (126 MHz, DMSO-d6) δ 160.72 (ArC═O), 158.76 (ArC), 152.82 (ArC),146.11 (ArC), 145.80 (ArC), 136.84 (ArCH), 132.01 (ArC), 131.55 (ArCH),128.73 (ArCH), 128.08 (ArCH), 126.99 (ArCH), 116.96 (ArC), 105.49(ArCH), 104.83 (ArCH), 55.59 (OCH₃).

MS (EI⁺): 283.1.

Synthesis of 2-(3,4,5-trimethoxyphenyl)-4H-3,1-benzoxazin-4-one(Intermediate 1n)

Chemical Formula: C17H15NO5 Molecular Weight: 313.30

The synthetic procedure followed the general method above usinganthranilic acid (0.50 g, 3.65 mmol) dissolved in pyridine (5 ml) and2.2 equivalent of 3,4,5-dimethoxybenzoyl chloride (1.85 g, 8.02 mmol).Collected as a white solid, yield 91% (1.04 g), mp 165° C.

¹H NMR (500 MHz, DMSO-d6) δ 8.17 (dd, J=9.3, 1.8 Hz, 1H, ArH), 7.98-7.94(m, 1H, ArH), 7.75 (d, J=2.9 Hz, 1H, ArH), 7.63 (td, J=15.7, 1.2 Hz, 1H,ArH), 7.49 (s, 2H, ArH), 3.92 (s, 6H, OCH3), 3.80 (s, 3H, OCH₃).

¹³C NMR (126 MHz, Chloroform) δ 159.61 (ArC═O), 156.82 (ArC), 153.34(ArC), 147.07 (ArC), 142.21 (ArC), 136.58 (ArCH), 128.64 (ArCH), 128.09(ArCH), 127.09 (ArCH), 125.23 (ArC), 116.78 (ArC), 105.60 (ArCH), 61.01(OCH₃), 56.42 (OCH₃).

MS (EI⁺): 313.1.

Synthesis of 2-(3,5-difluorophenyl)-4H-3,1-benzoxazin-4-one(Intermediate 1o)

Chemical Formula: C14H7F2NO2 Molecular Weight: 259.21

The synthetic procedure followed the general method above usinganthranilic acid (0.50 g, 3.65 mmol) dissolved in pyridine (5 ml) and2.2 equivalent of 3,5-difluorobenzoyl chloride (0.95 ml, 8.02 mmol).Collected as a white solid, yield 94% (0.89 g), mp 122° C.

¹H NMR (500 MHz, Chloroform) δ 8.28 (dd, J=9.4, 1.6 Hz, 1H, ArH), 7.88(m, 3H, ArH), 7.74 (dd, 8.1, 0.6 Hz, 1H, ArH), 7.60 (tt, 15.3, 1.4 Hz,1H, ArH), 7.05 (m, 1H, ArH).

¹³C NMR (126 MHz, Chloroform) δ 164.11 (d, J_(C-F)=10.1 Hz, ArC—F),162.12 (d, J_(C-F)=18.1 Hz, ArC—F), 158.78 (ArC═O), 146.35 (ArC), 136.78(ArCH), 133.75 (ArC), 128.98 (ArCH), 128.78 (ArCH), 127.49 (ArCH),117.14 (ArC), 111.40 (d, J_(C-F)=7.7 Hz, ArCH), 111.23 (d, J_(C-F)=7.2Hz, ArCH), 107.93 (ArCH), 105.93 (ArC).

MS (EI⁺): 259.1.

Synthesis of 2-(2,6-difluorophenyl)-4H-3,1-benzoxazin-4-one(Intermediate 1p)

Chemical Formula: C14H7F2NO2 Molecular Weight: 259.21

The synthetic procedure followed the general method above usinganthranilic acid (0.50 g, 3.65 mmol) dissolved in pyridine (5 ml) and2.2 equivalent of 2,6-difluorobenzoyl chloride (1.01 ml, 8.02 mmol).Collected as a white solid, yield 97% (0.92 g), mp 119° C.

¹H NMR (500 MHz, Chloroform-d) δ 8.31 (dd, J=7.8, 2.0 Hz, 1H, ArH),7.92-7.88 (m, 1H, ArH), 7.76 (dd, J=8.1, 1.7 Hz, 1H, ArH), 7.64 (td,J=7.3, 1.2 Hz, 1H, ArH), 7.52 (tt, J=8.5, 6.2 Hz, 1H, ArH), 7.08 (tt,J=7.1, 4.5 Hz, 2H, ArH).

¹³C NMR (126 MHz, DMSO-d6) δ 161.85 (d, J_(C-F)=6.2 Hz, ArC—F), 159.75(d, J_(C-F)=7.4 Hz, ArC—F), 158.91 (C═O), 150.95 (ArC), 146.19 (ArC),136.68 (ArCH), 133.55 (ArC), 133.03 (t, J_(C-F)=21.1 Hz, ArCH), 129.38(ArCH), 128.68 (ArCH), 127.49 (ArCH), 117.26 (ArC), 112.25 (d,J_(C-F)=4.2 Hz, ArCH), 112.07 (d, J_(C-F)=4.4 Hz, ArCH).

MS (EI⁺): 259.1.

Synthesis of 2-(2,4-difluorophenyl)-4H-3,1-benzoxazin-4-one(Intermediate 1q)

Chemical Formula: C14H7F2NO2 Molecular Weight: 259.21

The synthetic procedure followed the general method above usinganthranilic acid (0.50 g, 3.65 mmol) dissolved in pyridine (5 ml) and2.2 equivalent of 2,4-difluorobenzoyl chloride (0.99 ml, 8.02 mmol).Collected as a white solid, yield 81% (0.76 g), mp 102° C.

¹H NMR (500 MHz, Chloroform-d) δ 8.28 (dd, J=7.9, 0.6 Hz, 1H, ArH), 8.20(td, J=8.6, 6.4 Hz, 1H, ArH), 7.91-7.84 (m, 1H, ArH), 7.73 (dd, J=8.2,0.6 Hz, 1 Hz, ArH), 7.59 (td, J=7.8, 1.2 Hz, 1H, ArH), 7.08 (m, 2H,ArH).

¹³C NMR (126 MHz, DMSO-d6) δ 164.50 (d, J_(C-F)=252.9 Hz, ArC—F), 162.20(d, J_(C-F)=289.0 Hz, ArC—F), 158.99 (ArC═O), 146.63 (ArC), 136.68(ArCH), 132.85 (ArCH), 128.78 (ArCH), 128.62 (ArCH), 127.48 (ArCH),116.97 (ArC), 113.95 (ArC), 112.00 (d, J_(C-F)=21.9 Hz), 107.25 (ArC),105.63 (ArCH).

MS (EI⁺): 259.0.

Step 2—Synthesis of Example Compounds Synthesis of2-[(2-fluorobenzoyl)amino]-N-(2-morpholin-4-ylethyl)benzamide (Compound1a)

Chemical Formula: C20H22FN3O3 Molecular Weight: 371.41

The synthetic procedure followed the general method for step 2 aboveusing Intermediate 1a (1.00 g, 4.15 mmol) in DMF (10 ml), 2 equivalentsof DIPEA (1.19 ml, 8.29 mmol) and 2.2 equivalents of2-morpholinoethanamine (1.44 ml, 9.12 mmol). The product wasrecrystallized from ethanol as a white solid. Yield 47% (0.73 g), mp131° C.

¹H NMR (500 MHz, DMSO-d6) δ 12.00 (s, 1H, NH), 8.72 (s, 1H, NH), 8.57(d, J=8.3 Hz, 1H, ArH), 7.89 (td, J=7.7, 1.9 Hz, 1H, ArH), 7.76 (dd,J=8.0, 1.5 Hz, 1H, ArH), 7.68-7.64 (m, 1H, ArH), 7.57 (td, J=8.6, 1.5Hz, 1H, ArH), 7.46-7.35 (m, 2H, ArH), 7.24 (td, J=7.6, 1.2 Hz, 1H, ArH),3.53 (t, J=4.6 Hz, 4H, CH₂), 3.40 (q, J=6.4 Hz, 2H, CH₂), 2.51 (t, J=6.7Hz, 2H, CH₂), 2.47 (t, J=4.7 Hz, 4H, CH₂).

¹³C NMR (126 MHz, DMSO-d6) δ 168.03 (C═O), 160.86 (d, J_(C-F)=146.2 Hz,ArC—F), 158.28 (C═O), 138.32 (ArC), 133.77 (d, J_(C-F)=8.8 Hz, ArCH),131.83 (ArCH), 130.62 (d, J_(C-F)=1.3 Hz, ArCH), 128.02 (ArCH), 125.04(d, J_(C-F)=3.8 Hz, ArCH), 123.30 (ArCH), 122.94 (d, J_(C-F)=13.9 Hz,ArC) 121.73 (ArC), 120.96 (ArCH), 116.57 (d, J_(C-F)=22.7 Hz, ArCH),66.15 (CH₂), 56.98 (CH₂), 53.18 (CH₂), 36.43 (CH₂).

MS (ES+): 372.19 [M+1].

Synthesis of Hydrochloride Salt of Compound 1a

Compound 1a (0.31 g, 0.81 mmol) was dissolved in 150 ml of methanol.Hydrogen chloride in methanol (1.2 ml, 1.25M) was added and the mixturewas stirred at r.t. for an hour. Methanol was evaporated from themixture, followed by addition and evaporation of hexane. DCM (2 ml) wasadded to the mixture followed by addition of hexane and the formed whiteprecipitate was filtered under reduced pressure. Yield 79%, (0.26 g), mp145° C.

¹H NMR (500 MHz, Chloroform) δ 11.61 (s, 1H, NH), 9.00 (s, 1H, NH), 8.65(d, J=8.2 Hz, 1H, ArH), 8.00 (td, J=7.6, 2.0 Hz, 2H, ArH), 7.50-7.43 (m,2H, ArH), 7.25 (t, J=7.5 Hz, 1H, ArH), 7.15 (t, J=5.3 Hz, 1H, ArH), 7.12(d, J=7.0 Hz, 1H, ArH), 4.06 (t, J=12.2 Hz, 2H, CH₂), 3.93-3.78 (m, 4H,CH₂) 3.56 (d, J=11.8 Hz, 2H, CH₂), 3.20 (s, 2H, CH₂), 3.01 (s, 1H, NH⁺),2.87 (t, J=8.3, 7.6 Hz, 2H, CH₂).

¹³C NMR (126 MHz, DMSO-d6) δ 169.51 (C═O), 162.15 (ArC—F), 162.16 (ArC),161.23 (ArC), 159.24 (ArC), 139.50 (ArC), 133.25 (d, J_(C-F)=8.8 Hz,ArC), 132.85 (ArCH), 131.70 (d, J_(C-F)=2.2 Hz, ArCH), 128.40 (ArCH),124.69 (d, J_(C-F)=3.4 Hz, ArCH), 123.73 (ArCH), 122.04 (ArCH), 120.17(ArC), 116.35 (d, J_(C-F)=23.9 Hz, ArCH), 63.53 (CH₂), 58.72 (CH₂),53.11 (CH₂), 34.00 (CH₂).

Calculated analysis for C20H22FN3O3 (371.41): C, 64.68; H, 5.97; N,11.31. Found C, 64.49; H, 6.08; N, 11.29.

MS (EI⁺): 372.2

Synthesis of2-[(4-fluorobenzoyl)amino]-N-(2-morpholin-4-ylethyl)benzamide (Compound1c)

Chemical Formula: C20H22FN3O3 Molecular Weight: 371.41

The synthetic procedure followed the general method for step 2 aboveusing Intermediate 1c (0.50 g, 2.06 mmol) in DMF (8 ml), 2 equivalentsof DIPEA (0.72 ml, 4.12 mmol) and 2.2 equivalents of2-morpholinoethanamine (0.60 ml, 4.54 mmol). The product wasrecrystallized from ethanol as a white solid. Yield 8% (0.05 g), mp 129°C.

¹H NMR (500 MHz, DMSO-d6) δ 12.52 (s, 1H, NH), 8.80 (s, 1H, NH), 8.61(d, J=8.3 Hz, 1H, ArH), 8.0 (td, J=14.1, 2.1 Hz, 2H, ArH), 7.83 (dd,J=7.9, 1.5 Hz, 1H, ArH), 7.58 (td, J=8.2, 7.8, 1.5 Hz, 1H, ArH), 7.45(t, J=17.6 Hz, 2H, ArH), 7.23 (t, J=7.6, 1 H, ArH), 3.55 (t, J=4.6 Hz,4H, CH₂), 3.44 (q, J=6.5 Hz, 2H, CH₂), 2.51 (q, J=6.3 Hz, 6H, CH₂).

¹³C NMR (126 MHz, DMSO-d6) δ 168.50 (C═O), 164.29 (d, J_(C-F)=253.3 Hz,ArC—F), 158.31 (C═O), 139.17 (ArC), 132.15 (ArCH), 131.05 (d,J_(C-F)=3.8 Hz, ArC), 129.61 (d, J_(C-F)=10.1 Hz, ArCH), 128.10 (ArCH),122.94 (ArCH), 120.58 (ArC), 120.38 (ArCH), 115.98 (d, J_(C-F)=20.2 Hz,ArCH), 66.17 (CH₂), 56.96 (CH₂), 53.20 (CH₂), 36.55 (CH₂).

MS (ES+): 372.20 [M+1].

Calculated analysis for C20H22FN3O3 (371.41): C, 64.62; H, 6.09; N,11.32. Found C, 64.49; H, 6.08; N, 11.29.

Synthesis of2-[(3-methoxybenzoyl)amino]-N-(2-morpholin-4-ylethyl)benzamide (Compound1e)

Chemical Formula: C21H25N3O4 Molecular Weight: 383.44

The synthetic procedure followed the general method for step 2 aboveusing Intermediate 1e (0.50 g, 1.97 mmol) in DMF (8 ml), 2 equivalentsof DIPEA (0.69 ml, 3.94 mmol) and 2.2 equivalents of2-morpholinoethanamine (0.57 ml, 4.34 mmol). The product wasrecrystallized from ethanol as a white solid. Yield 0.33 g (44%), mp105° C.

¹H NMR (500 MHz, DMSO-d6) δ 12.49 (s, 1H, NH), 8.80 (s, 1H, NH), δ 8.63(d, J=8.5 Hz, 1H, ArH), 7.82 (dd, J=7.9, 1.6 Hz, 1H, ArH), 7.58 (td,J=17.0, 1.2 Hz, 1H, ArH), 7.52 (d, J=6.5, Hz, 2H, ArH), 7.48 (t, J=3.2Hz, 1H, ArH), 7.25-7.16 (m, 2H, ArH), 3.86 (s, 3H, CH₃), 3.54 (t, J=4.7Hz, 4H, CH₂), 3.44 (q, J=6.5 Hz, 2H, CH₂), 2.50 (d, J=15.1 Hz, 2H, CH₂),2.42 (t, J=4.7 Hz, 4H, CH₂).

¹³C NMR (126 MHz, DMSO-d6) δ 168.50 (C═O), 164.09 (C═O), 159.52 (ArC),139.19 (ArC), 136.01 (ArC), 132.14 (ArCH), 130.13 (ArCH), 128.10 (ArCH),122.87 (ArCH), 120.55 (ArC), 120.27 (ArCH), 118.80 (ArCH), 117.63(ArCH), 112.46 (ArCH), 66.17 (CH₂), 56.99 (CH₂), 55.28 (0 CH₃), 53.18(CH₂), 36.53 (CH₂).

MS (ES+): 384.19 [M+1].

Calculated analysis for C21H25N3O4 (383.44): C, 65.78; H, 6.57; N,10.95. Found C, 65.71; H, 6.77; N, 10.98.

Synthesis of2-[(4-methoxybenzoyl)amino]-N-(2-morpholin-4-ylethyl)benzamide (Compound1f)

Chemical Formula: C21H25N3O4 Molecular Weight: 383.44

The synthetic procedure followed the general method for step 2 aboveusing Intermediate if (0.50 g, 1.97 mmol) in DMF (8 ml), 2 equivalentsof DIPEA (0.69 ml, 3.94 mmol) and 2.2 equivalents of2-morpholinoethanamine (0.57 ml, 4.34 mmol). The product wasrecrystallized from ethanol as a white solid. Yield 32% (0.24 g), mp110° C.

¹H NMR (500 MHz, DMSO-d6) δ 12.44 (s, 1H, NH), 8.78 (s, 1H, NH), 8.65(d, J=8.3 Hz, 1H, ArH), 7.92 (d, J=8.9 Hz, 2H, ArH), 7.81 (dd, J=8.0,1.5 Hz, 1H, ArH), 7.56 (td, J=8.7, 1.5 Hz, 1H, ArH), 7.19 (d, J=15.5,1.3 Hz, 3H, ArH), 3.86 (s, 3H, OCH₃), 3.55 (t, J=4.6 Hz, 4H, CH₂), 3.45(q, J=6.4 Hz, 2H, CH₂), 2.51 (m, 3H, CH₂), 2.43 (d, J=9.1 Hz, 3H, CH₂).

¹³C NMR (126 MHz, DMSO-d6) δ 168.60 (C═O), 163.87 (C═O), 162.20 (ArC),140.73 (ArC), 132.10 (ArCH), 128.80 (ArCH), 128.06 (ArCH), 126.63 (ArC),122.50 (ArCH), 120.23 (ArC), 120.18 (ArCH), 114.18 (ArCH), 66.18 (CH₂),56.98 (CH₂), 55.45 (OCH₃), 53.21 (CH₂), 36.54 (CH₂).

MS (ES+): 384.19 [M+1].

Calculated analysis for C21H25N3O4 (383.44): C, 65.78; H, 6.57; N,10.95. Found C, 65.63; H, 6.62; N, 10.95.

Synthesis of2-[(2-nitrobenzoyl)amino]-N-(2-morpholin-4-ylethyl)benzamide (Compound1g)

Chemical Formula: C20H22N4O5 Molecular Weight: 398.16

The synthetic procedure followed the general method for step 2 aboveusing Intermediate 1 g (0.50 g, 1.87 mmol) in DMF (8 ml), 2 equivalentsof DIPEA (0.65 ml, 3.74 mmol) and 2.2 equivalents of2-morpholinoethanamine (0.54 ml, 4.11 mmol). The product wasrecrystallized from ethanol as a white solid. Yield 49% (0.37 g), mp139° C.

¹H NMR (500 MHz, DMSO-d6) δ 12.02 (s, 1H, NH), 8.75 (s, 1H, NH), 8.41(d, J=8.2 Hz, 1H, ArH), 8.13 (d, J=8.3, 1H, ArH), 7.91 (t, J=15.2 Hz,1H, ArH), 7.82 (m, 3H, ArH), 7.59 (t, J=8.2 Hz, 1H, ArH), 7.27 (td,J=7.6, 1.3 Hz, 1H, ArH), 3.53 (t, J=4.6 Hz, 4H, CH₂), 3.37 (q, J=6.5 Hz,2H, CH₂), 2.46 (t, J=6.8 Hz, 2H, CH₂), 2.39 (t, J=4.7 Hz, 4H, CH₂)

¹³C NMR (126 MHz, DMSO-d6) δ 168.01 (C═O), 163.35 (C═O), 147.02 (ArC),138.23 (ArC), 134.11 (ArCH), 132.02 (ArC), 131.58 (ArCH), 131.68 (ArCH),128.38 (ArCH), 128.11 (ArCH), 124.57 (ArCH), 123.67 (ArCH), 121.76(ArC), 120.89 (ArCH), 66.14 (CH₂), 56.95 (CH₂), 53.19 (CH₂), 36.48(CH₂).

MS (ES+): 399.19 [M+1].

Calculated analysis for C20H22N4O5 (398.16): C, 60.29; H, 5.57; N,14.06. Found C, 60.34; H, 5.61; N, 13.97.

Synthesis of 2-[4-nitrobenzoyl)amino]-N-(2-morpholin-4-ylethyl)benzamide (Compound 1i)

Chemical Formula: C20H22N4O5 Molecular Weight: 398.16

The synthetic procedure followed the general method for step 2 aboveusing Intermediate 1i (0.50 g, 1.87 mmol) in DMF (8 ml), 2 equivalentsof DIPEA (0.65 ml, 3.74 mmol) and 2.2 equivalents of2-morpholinoethanamine (0.54 ml, 4.11 mmol). The product wasrecrystallized from ethanol as a white solid. Yield 51% (0.38 g), mp148° C.

¹H NMR (500 MHz, DMSO-d6) δ 12.70 (s, 1H, NH), 8.83 (s, 1H, NH), 8.59(d, J=8.4 Hz, 1H, ArH), 8.44 (d, 8.9 Hz, 2H, ArH), 8.16 (d, 8.9 Hz, 2H,ArH), 7.85 (dd, J=8.0, 1.5 Hz, 1H, ArH), 7.60 (td, J=8.6, 1.5 Hz, 1H,ArH), 7.27 (td, J=7.5, 1.2 Hz, 1H, ArH), 3.54 (t, J=4.6 Hz, 4H, CH₂),3.44 (q, J=6.5 Hz, 2H, CH₂), 2.44 (m, 6H, CH₂).

¹³C NMR (126 MHz, DMSO-d6) δ 168.38 (C═O), 162.69 (C═O), 149.41 (ArC),140.10 (ArC), 138.76 (ArC), 132.21 (ArCH), 128.43 (ArCH), 128.15 (ArCH),124.13 (ArCH), 123.47 (ArCH), 120.92 (ArC), 120.64 (ArCH), 66.17 (CH₂),56.93 (CH₂), 53.20 (CH₂), 36.58 (CH₂).

MS (ES+): 399.21 [M+1].

Calculated analysis for C20H22N4O5 (398.16): C, 60.29; H, 5.57; N,14.06. Found C, 60.36; H, 5.54; N, 14.05.

Synthesis of 2-[(2-methylbenzoyl)amino]-N-(2-morpholin-4-ylethyl)benzamide (Compound 1j)

Chemical Formula: C21H25N3O3 Molecular Weight: 367.44

The synthetic procedure followed the general method for step 2 aboveusing Intermediate 1j (0.50 g, 2.11 mmol) in DMF (8 ml), 2 equivalentsof DIPEA (0.61 ml, 4.64 mmol) and 2.2 equivalents of2-morpholinoethanamine (0.74 ml, 4.22 mmol). The product wasrecrystallized from ethanol as a white solid. Yield 14% (0.11 g), mp 76°C.

¹H NMR (500 MHz, DMSO-d6) δ 11.78 (s, 1H, NH), 8.72 (s, 1H, NH), 8.59(d, J=8.3 Hz, 1H, ArH), 7.78 (dd, J=7.8, 1.5 Hz, 1H, ArH), 7.56 (q,J=23.6 Hz, 2H, ArH), 7.43 (td, J=7.5, 1.4 Hz, 1H, ArH), 7.34 (dt, J=7.3,3.4 Hz, 2H, ArH), 7.22 (td, J=7.6, 1.2 Hz, 1H, ArH), 3.53 (t, J=4.6 Hz,4H, CH₂), 3.37 (q, J=6.5 Hz, 2H, CH₂), 2.45 (d, J=3.9 Hz, 5H, CH_(2/3)),2.39 (t, J=4.7 Hz, 4H, CH₂).

¹³C NMR (126 MHz, DMSO-d6) δ 168.22 (C═O), 167.10 (C═O), 138.96 (ArC),136.44 (ArC), 135.82 (ArC), 131.96 (ArCH), 131.14 (ArCH), 130.24 (ArCH),128.04 (ArCH), 126.75 (ArCH), 126.07 (ArCH), 122.92 (ArCH), 121.00(ArC), 120.32 (ArCH), 66.14 (CH₂), 56.99 (CH₂), 53.19 (CH₂), 36.43(CH₂), 19.59 (CH₃).

MS (ES+): 368.22 [M+1].

Calculated analysis for C21H25N3O3 (367.44): C, 68.64; H, 6.86; N,11.44. Found C, 68.27; H, 6.71; N, 11.28.

Synthesis of 3,4-dimethoxy-N-(2-[(2-morpholin-4-ylethyl) carbamoyl)phenyl) benzamide (Compound 11)

Chemical Formula: C22H27N3O5 Molecular Weight: 413.47

The synthetic procedure followed the general method for step 2 aboveusing Intermediate 1l (0.25 g, 0.88 mmol) in DMF (8 ml), 2 equivalentsof DIPEA (0.31 ml, 1.77 mmol) and 2.2 equivalents of2-morpholinoethanamine (0.26 ml, 1.94 mmol). The product wasrecrystallized from ethanol as a white solid. Yield 22% (0.19 g), mp106° C.

¹H NMR (500 MHz, DMSO-d6) δ 12.47 (s, 1H, NH), 8.79 (s, 1H, NH), 8.64(d, J=8.4 Hz, 1H, ArH), 7.81 (d, J=7.8 Hz, 1H, ArH), 7.60-7.50 (m, 3H,ArH), 7.23-7.13 (m, 2H, ArH), 3.86 (s, 6H, OCH₃), 3.55 (t, J=4.4 Hz, 4H,CH₂), 3.45 (q, J=6.5 Hz, 2H, CH₂), 2.42 (t, J=4.6 Hz, 6H, CH₂).

¹³C NMR (126 MHz, DMSO-d6) δ 168.62 (C═O), 163.98 (ArC), 151.93 (ArC),148.69 (ArC), 139.52 (ArC), 132.12 (ArCH), 128.06 (ArCH), 126.83 (ArC),122.50 (ArCH), 120.26 (ArC), 120.07 (ArCH), 119.86 (ArCH), 111.38(ArCH), 110.45 (ArCH), 66.17 (CH₂), 56.98 (CH₂), 55.70 (OCH₃), 55.45(OCH₃), 53.20 (CH₂), 36.52 (CH₂).

MS (EI+): 413.2.

Calculated analysis for C22H27N3O5 (413.47): C, 63.91; H, 6.58; N,10.16. Found C, 63.54; H, 6.86; N, 10.11

Synthesis of 3,5-dimethoxy-N-(2-[(2-morpholin-4-ylethyl)carbamoyl]phenyl) benzamide (Compound 1m)

Chemical Formula: C22H27N3O5 Molecular Weight: 413.47

The synthetic procedure followed the general method for step 2 aboveusing Intermediate 1m (0.25 g, 0.88 mmol) in DMF (8 ml), 2 equivalentsof DIPEA (0.31 ml, 1.77 mmol) and 2.2 equivalents of2-morpholinoethanamine (0.26 ml, 1.94 mmol). The product wasrecrystallized from ethanol as a white solid. Yield 58% (0.21 g), mp120° C.

¹H NMR (500 MHz, DMSO-d6) δ 12.46 (s, 1H, NH), 8.79 (s, NH), 8.61 (dd,J=8.4, 1.2 Hz, 1H, ArH), 7.81 (dd, J=7.9 Hz, 1H, ArH), 7.57 (t, J=7.6Hz, 1H, ArH), 7.22 (td, J=7.5, 1.3 Hz, 1H, ArH), 7.07 (d, J=2.2 Hz, 2H,ArH), 6.77 (t, J=2.2 Hz, 1H, ArH), 3.84 (d, J=1.5 Hz, 6H, OCH₃), 3.54(t, J=4.6 Hz, 4H, CH₂), 3.44 (q, J=6.4 Hz, 2H, CH₂), 2.49 (t, J=6.8 Hz,2H, CH₂), 2.42 (t, J=4.7 Hz, 4H, CH₂)

¹³C NMR (126 MHz, DMSO-d6) δ 168.49 (C═O), 163.95 (ArC), 160.70 (ArC),139.12 (ArC), 136.72 (ArC), 132.13 (ArCH), 128.09 (ArCH), 122.89 (ArCH),120.59 (ArC), 120.22 (ArCH), 104.99 (ArCH), 103.43 (ArCH), 66.16 (CH₂),56.99 (CH₂), 55.44 (OCH₃), 53.21 (CH₂), 36.52 (CH₂).

MS (EI+): 413.2.

Calculated analysis for C22H27N3O5 (413.47): C, 63.91; H, 6.58; N,10.16. Found C, 63.66; H, 6.43; N, 10.03.

Synthesis of 3,4,5-trimethoxy-N-(2-[(2-morpholin-4-ylethyl)carbamoyl]phenyl) benzamide (Compound 1n)

Chemical Formula: C23H29N3O6 Molecular Weight: 443.21

The synthetic procedure followed the general method for step 2 aboveusing Intermediate 1n (0.25 g, 0.80 mmol) in DMF (8 ml), 2 equivalentsof DIPEA (0.28 ml, 1.60 mmol) and 2.2 equivalents of2-morpholinoethanamine (0.23 ml, 1.76 mmol). The product wasrecrystallized from ethanol as a white solid. Yield 52% (0.18 g), mp117° C.

¹H NMR (500 MHz, DMSO-d6) δ 11.78 12.49 (s, 1H, NH), 8.80 (s, 1H, NH),8.59 (dd, J=8.3, 1.3 Hz, 1H, ArH), 7.81 (dd, J=7.9, 1.5 Hz, 1H, ArH),7.57 (td, J=8.8, 2.0 Hz, 1H, ArH), 7.26 (s, 2H, ArH), 7.21 (td, J=8.8,1.4 Hz, 1H, ArH), 3.89 (s, 6H, OCH₃), 3.77 (s, 3H, OCH₃), 3.54 (t, J=4.7Hz, 4H, CH₂), 3.43 (q, J=6.5 Hz, 2H, CH₂), 2.49 (d, J=6.8 Hz, 2H, CH₂),2.41 (t, J=7.3 Hz, 4H, CH₂).

¹³C NMR (126 MHz, DMSO-d6) δ 168.54 (C═O), 163.92 (ArC), 152.90 (ArC),140.63 (ArC), 139.25 (ArC), 132.12 (ArCH), 129.91 (ArC), 128.08 (ArCH),122.77 (ArCH), 120.62 (ArC), 120.14 (ArCH), 104.56 (ArCH), 66.15 (CH₂),60.13 (OCH₃), 56.99 (CH₂), 55.94 (OCH₃), 53.20 (CH₂), 36.49 (CH₂).

MS (EI+): 443.2.

Calculated analysis for C23H29N3O6 (443.21): C, 62.29; H, 6.59; N, 9.47.Found C, 62.29; H, 6.46; N, 9.49.

Synthesis of 3,5-difluoro-N-(2-[(2-morpholin-4-ylethyl)carbamoyl]phenyl) benzamide (Compound 1o)

Chemical Formula: C20H21F2N3O3 Molecular Weight: 389.40

The synthetic procedure followed the general method for step 2 aboveusing Intermediate 10 (0.25 g, 0.96 mmol) in DMF (8 ml), 2 equivalentsof DIPEA (0.33 ml, 1.92 mmol) and 2.2 equivalents of2-morpholinoethanamine (0.28 ml, 2.11 mmol). The product wasrecrystallized from ethanol as a white solid. Yield 40% (0.15 g), mp116° C.

¹H NMR (500 MHz, DMSO-d6) δ 12.54 (s, 1H, NH), 8.81 (s, 1H, NH), 8.52(dd, J=8.4, 1.2 Hz, 1H, ArH), 7.83 (dd, J=7.9, 1.5 Hz, 1H, ArH),7.61-7.59 (m, 1H, ArH), 7.59-7.55 (m, 3H, ArH), 7.26 (td, J=7.6, 1.3 Hz,2H, ArH), 3.55 (t, J=4.6 Hz, 4H, CH₂), 3.44 (q, J=6.1 Hz, 2H, CH₂), 2.49(d, J=6.8 Hz, 1H, CH₂), 2.42 (t, J=6.5 Hz, 4H, CH₂).

¹³C NMR (126 MHz, DMSO-d6) δ 168.33 (C═O), 163.50 (d, J_(C-F)=13.1 Hz,ArC—F), 161.50 (d, J_(C-F)=11.2 Hz, ArC—F), 150.10 (ArC), 143.55 (ArC),138.5 (d, J_(C-F)=37.7 Hz, ArC), 132.15 (ArCH), 128.16 ArCH), 123.47(ArCH), 121.16 (ArC), 120.69 (ArCH), 110.50 (d, J_(C-F)=7.04 Hz, ArCH),110.29 (d, J_(C-F)=6.7 Hz, ArCH), 107.50 (ArCH), 66.15 (CH₂), 56.98(CH₂), 53.21 (CH₂), 36.56 (CH₂).

MS (EI+): 389.2.

Calculated analysis for C20H21F2N3O3 (389.40): C, 61.69; H, 5.44; N,10.79. Found C, 61.56; H, 5.27; N, 10.68.

Synthesis of 2,6-difluoro-N-(2-[(2-morpholin-4-ylethyl)carbamoyl]phenyl) benzamide (Compound 1p)

Chemical Formula: C20H21F2N3O3 Molecular Weight: 389.40

The synthetic procedure followed the general method for step 2 aboveusing Intermediate 1p (0.25 g, 0.96 mmol) in DMF (8 ml), 2 equivalentsof DIPEA (0.33 ml, 1.92 mmol) and 2.2 equivalents of2-morpholinoethanamine (0.28 ml, 2.11 mmol). The product wasrecrystallized from ethanol as a white solid. Yield 61% (0.25 g), mp136° C.

¹H NMR (500 MHz, DMSO-d6) δ 12.03 (s, 1H, NH), 8.76 (s, 1H, NH), 8.52(dd, J=8.3, 1.2 Hz, 1H, ArH), 7.79 (dd, J=8.0, 1.6 Hz, 1H, ArH),7.66-7.62 (m, 1H, ArH), 7.62-7.57 (m, 1H, ArH), 7.28 (td, J=7.9, 6.2 Hz,3H, ArH), 3.53 (t, J=4.7 Hz, 4H, CH₂), 3.37 (q, J=6.4 Hz, 2H, CH₂), 2.46(t, J=6.8 Hz, 2H, CH₂), 2.39 (t, J=5.0 Hz, 4H, CH₂).

¹³C NMR (126 MHz, DMSO-d6) δ 167.96 (C═O), 159.76 (d, J_(C-F)=7.1 Hz,ArC—F), 157.72 (d, J_(C-F)=5.5 Hz, ArC—F), 137.98 (ArC), 132.79 (t,J_(C-F)=20.4 Hz, ArCH), 132.13 (ArCH), 128.14 (ArCH), 127.75 (ArC),123.77 (ArCH), 121.32 (ArC), 120.54 (ArCH), 112.52 (d, J_(C-F)=3.8 Hz,ArCH), 112.34 (d, J_(C-F)=4.2 Hz, ArCH), 111.50 (ArC), 66.13 (CH₂),56.92 (CH₂), 53.18 (CH₂), 36.45 (CH₂).

MS (EI+): 389.2.

Calculated analysis for C20H21F2N3O3 (389.40): C, 61.69; H, 5.44; N,10.79. Found C, 61.68; H, 5.31; N, 10.82.

Synthesis of 2,4-difluoro-N-(2-[(2-morpholin-4-ylethyl)carbamoyl]phenyl) benzamide (Compound 1q)

Chemical Formula: C20H21F2N3O3 Molecular Weight: 389.40

The synthetic procedure followed the general method for step 2 aboveusing Intermediate 1q (0.25 g, 0.96 mmol) in DMF (8 ml), 2 equivalentsof DIPEA (0.33 ml, 1.92 mmol) and 2.2 equivalents of2-morpholinoethanamine (0.28 ml, 2.11 mmol). The product wasrecrystallized from ethanol as a white solid. Yield 55% (0.21 g), mp121° C.

¹H NMR (500 MHz, DMSO-d6) δ 12.00 (s, 1H, NH), 8.72 (s, 1H, NH), 8.55(d, J=8.3 Hz, 1H, ArH), 7.98 (tt, J=8.8, 6.6 Hz, 1H, ArH), 7.76 (dd,J=7.9, 1.6 Hz, 1H, ArH), 7.57 (td, J=8.5, 1.5 Hz, 1H, ArH), 7.49 (td,J=11.6, 2.5 Hz, 1H, ArH), 7.30 (td, J=8.4, 3.8 Hz, 1H, ArH), 7.24 (td,J=7.6, 1.2 Hz, 1H, ArH), 3.53 (t, J=5.6 Hz, 4H, CH₂), 3.39 (q, J=6.3 Hz,2H, CH₂), 2.48 (t, J=6.7 Hz, 2H, CH₂), 2.41 (t, J=4.6 Hz, 4H, CH₂).

¹³C NMR (126 MHz, DMSO-d6) δ 168.02 (C═O), 163.20 (d, J_(C-F)=5.3 Hz,ArC—F), 158.10 (d, J_(C-F)=10.5 Hz, ArC—F), 138.24 (d, J_(C-F)=5.4 Hz,ArC), 132.69 (d, J_(C-F)14.9 Hz, ArC), 131.83 (ArCH), 128.02 (ArCH),123.38 (ArCH), 121.80 (ArC), 121.04 (ArCH), 119.75 (ArC), 112.57 (d,J_(C-F)=3.3 Hz, ArCH), 112.41 (d, J_(C-F)=3.7 Hz, ArCH), 104.99 (ArCH),66.16 (CH₂), 56.98 (CH₂), 53.18 (CH₂), 36.44 (CH₂).

MS (EI+): 389.2.

Calculated analysis for C20H21F2N3O3 (389.40): C, 61.69; H, 5.44; N,10.79. Found C, 61.59; H, 5.24; N, 10.70.

Example 2 Synthesis of Compounds of Series 2

The compounds of series 2 have the general formula

Compound R¹ 2a

2b

2c

2d

2e

2f —NH₂

Step 1—Synthesis of Example Compounds Synthesis of2-[(2-fluorobenzoyl)amino]-N-2-morpholin-4-ylpropyl)benzamide (Compound2a)

Chemical Formula: C21H24FN3O3 Molecular Weight: 385.43

The synthetic procedure followed the general method for step 2 aboveusing Intermediate 1a (0.50 g, 2.07 mmol) in DMF (8 ml), 2 equivalentsof DIPEA (0.72 ml, 4.14 mmol) and 2.2 equivalents of3-morpholinopropan-1-amine (0.67 ml, 4.56 mmol). The product wasrecrystallized from ethanol as a white solid. Yield 17% (0.14 g), mp108° C.

¹H NMR (500 MHz, DMSO-d6) δ 12.06 (s, 1H, NH), 8.82 (s, 1H, NH), 8.58(d, J=8.3 Hz, 1H, ArH), 7.89 (td, J=7.7, 1.9 Hz, 1H, ArH), 7.77 (dd,J=7.9, 1.5 Hz, 1H, ArH), 7.69-7.61 (m, 1H, ArH), 7.57 (td, J=8.6, 1.5Hz, 1H, ArH), 7.44-7.36 (m, 2H, ArH), 7.23 (td, J=7.6, 1.2 Hz, 1H, ArH),3.55 (t, J=4.6 Hz, 4H, CH₂), 3.30 (dd, J=7.0 Hz, 2H, CH₂), 2.32 (t,J=6.6 Hz, 6H, CH₂), 1.69 (p, J=7.0 Hz, 2H, CH₂).

¹³C NMR (126 MHz, DMSO-d6) δ 168.01 (C═O), 160.86 (d, J_(C-F)=146.2 Hz,ArC—F), 158.29 (C═O), 138.37 (ArC), 133.76 (d, J_(C-F)=8.8 Hz, ArCH),131.78 (ArCH), 130.64 (d, J_(C-F)=1.3 Hz, ArCH), 128.01 (ArCH), 125.01(d, J_(C-F)=3.8 Hz, ArCH), 123.24 (ArCH), 122.91 (ArC), 121.70 (ArC),120.94 (ArCH), 116.64 (d, J_(C-F)=22.9 Hz, ArCH), 66.16 (CH₂), 55.99(CH₂), 53.30 (CH₂), 37.75 (CH₂), 25.59 (CH₂).

MS (ES+): 386.18 [M+1].

Calculated analysis for C21H24FN3O3 (385.43): C, 65.44; H, 6.28; N,10.09. Found C, 65.44; H, 6.39; N, 10.94.

Synthesis of 2-[(4-fluorobenzoyl)amino]-N-(pyridin-3-ylmethyl)benzamide(Compound 2b)

Chemical Formula: C21H18FN3O2 Molecular Weight: 363.38

The synthetic procedure followed the general method for step 2 aboveusing Intermediate 1a (0.50 g, 2.07 mmol) in DMF (8 ml), 2 equivalentsof DIPEA (0.72 ml, 4.14 mmol) and 2.2 equivalents of 2-(pyridin-2-yl)ethanamine (0.55 ml, 4.56 mmol). The product was recrystallized fromethanol as a brown solid. Yield 44% (0.34 g), mp 88° C.

¹H NMR (500 MHz, DMSO-d6) δ 11.99 (s, 1H, NH), 8.89 (s, 1H, NH), 8.57(d, J=8.4 Hz, 1H, ArH), 8.48 (d, J=6.6 Hz, 1H, ArH), 7.88 (td, J=7.8,1.8 Hz, 1H, ArH), 7.72 (dd, J=7.8, 1.6 Hz, 1H, ArH), 7.68 (m, 2H, ArH),7.56 (td, J=17.4, 1.3 Hz, 1H, ArH), 7.41 (m, 2H, ArH), 7.27 (d, J=7.7Hz, 1H, ArH), 7.22 (td, J=7.6, 1.3 Hz, 1H, ArH), 7.16 (td, J=7.5, 1.2Hz, 1H, ArH), 3.63 (m, 2H, CH₂), 3.00 (t, J=7.2 Hz, 2H, CH₂).

¹³C NMR (126 MHz, DMSO-d6) δ 168.03 (C═O), 160.88 (d, J_(C-F)=144.9 Hz,ArC—F), 158.94 (ArC), 158.30 (ArC), 148.97 (ArCH), 138.37 (ArC), 136.33(ArCH), 133.76 (d, J_(C-F)=8.8 Hz, ArCH), 131.81 (ArCH), 130.60 (d,J_(C-F)=1.3 Hz, ArCH), 127.99 (ArCH), 125.02 (d, J_(C-F)=2.6 Hz, ArCH),123.23 (ArCH), 123.12 (ArCH), 121.60 (ArC), 121.42 (ArCH), 120.89(ArCH), 116.49 (d, J_(C-F)=22.7 Hz, ArCH), 36.98 (CH₂).

MS (ES+): 362.13 [M+1]. High resolution MS: 364.1456 [M−1]—CompositionC21H19FN3O2 (delta ppm 0.1) or C18H21F502 (delta ppm −0.1).

Synthesis of2[(4-fluorobenzoyl)amino]-N-(pyrrolidin-3-ylmethyl)benzamide (Compound2c)

Chemical Formula: C20H22FN3O2 Molecular Weight: 355.17

The synthetic procedure followed the general method for step 2 aboveusing Intermediate 1a (0.50 g, 2.07 mmol) in DMF (8 ml), 2 equivalentsof DIPEA (0.72 ml, 4.14 mmol) and 2.2 equivalents of 2-(pyrrolidin-1-yl)ethanamine (0.58 ml, 4.56 mmol). The product was recrystallized fromethanol as a yellow/brown solid. Yield 41% (0.30 g), mp 89° C.

¹H NMR (500 MHz, DMSO-d6) δ 12.04 (s, 1H, NH), 8.74 (s, 1H, NH), 8.58(d, J=8.4 Hz, 1H, ArH), 7.88 (td, J=7.7, 1.9 Hz, 1H, ArH), 7.77 (dd,J=7.9, 1.5 Hz, 1H, ArH), 7.66 (m, 1H, ArH), 7.57 (td, J=8.6, 1.5 Hz, 1H,ArH), 7.41 (m, 2H, ArH), 7.23 (td, J=7.6, 1.2 Hz, 1H, ArH), 3.38 (q,J=6.5 Hz, 2H, CH₂), 2.57 (t, J=6.8 Hz, 2H, CH₂), 2.47 (t, J=6.6, Hz, 4H,CH₂), 1.71-1.60 (m, 4H, CH₂).

¹³C NMR (126 MHz, DMSO-d6) δ 167.98 (C═O), 161.44 (d, J_(C-F)=144.9 Hz,ArC—F), 158.29 (C═O), 138.38 (ArC), 133.77 (d, J_(C-F)=8.6 Hz, ArCH),131.83 (ArCH), 130.60 (ArCH), 128.04 (ArCH), 125.03 (d, J_(C-F)=3.8 Hz,ArCH), 123.27 (ArCH), 122.91 (d, J_(C-F)=12.6 Hz, ArC), 121.59 (ArCH),120.93 (ArCH), 116.58 (d, J_(C-F)=22.7 Hz, ArC), 54.49 (CH₂), 53.56(CH₂), 38.58 (CH₂), 23.12 (CH₂).

MS (ES+): 356.15 [M+1].

Calculated analysis for C20H22FN3O2 (355.17): C, 67.59; H, 6.24; N,11.82. Found C, 67.66; H, 6.19; N, 11.67.

Synthesis of2-[(4-fluorobenzoyl)amino]-N-(piperidin-3-ylmethyl)benzamide (Compound2d)

Chemical Formula: C21H24FN3O2 Molecular Weight: 369.43

The synthetic procedure followed the general method for step 2 aboveusing Intermediate 1a (0.50 g, 2.07 mmol) in DMF (8 ml), 2 equivalentsof DIPEA (0.72 ml, 4.14 mmol) and 2.2 equivalents of 2-(piperidin-1-yl)ethanamine 16d (0.65 ml, 4.56 mmol). The product was recrystallized fromethanol as a white solid. Yield 51% (0.39 g), mp 94° C.

¹H NMR (500 MHz, DMSO-d6) δ 11.99 (s, 1H, NH), 8.69 (s, 1H, NH), 8.57(d, J=8.3 Hz, 1H, ArH), 7.88 (td, J=17.1, 1.7 Hz, 1H, ArH), 7.76 (d,J=9.1 Hz, 1H, ArH), 7.67-7.62 (m, 1H, ArH), 7.56 (t, J=16.4 Hz, 1H,ArH), 7.42-7.37 (m, 1H, ArH), 7.23 (t, J=15.4 Hz, 2H, ArH), 3.36 (q,J=19.6 Hz, 2H, CH₂), 2.43 (t, J=14.1 Hz, 2H, CH₂), 2.35 (t, J=18.6 Hz,4H, CH₂), 1.44 (q, J=22.6 Hz, 4H, CH₂), 1.34 (d, J=15.1 Hz, 2H, CH₂).

¹³C NMR (126 MHz, DMSO-d6) δ 167.99 (C═O), 160.89 (d, J_(C-F)=155.0 Hz,ArC—F), 158.31 (C═O), 154.28 (ArC), 138.39 (ArC), 133.76 (d, J_(C-F)=8.8Hz, ArCH), 131.77 (ArCH), 130.60 (d, J_(C-F)=1.8 Hz, ArCH), 128.01(ArCH), 125.00 (d, J_(C-F)=3.7 Hz, ArCH), 123.26 (ArCH), 121.81 (ArC),120.97 (ArCH), 116.56 (d, J_(C-F)=22.8 Hz, ArCH), 57.31 (CH₂), 53.96(CH₂), 36.87 (CH₂), 25.56 (CH₂), 23.99 (CH₂).

MS (EI+): 369.19.

Calculated analysis for C21H24FN3O2 (369.43): C, 68.27; H, 6.55; N,11.37. Found C, 67.95; H, 6.82; N, 11.44.

Synthesis of N-(2-aminoethyl)-2-(2-fluorobenzamido) benzamide (Compound2f)

Chemical Formula: C16H16FN3O2 Molecular Weight: 301.32

The synthetic procedure followed the general method for step 2 aboveusing Intermediate 1a (0.50 g, 2.07 mmol) in DMF (8 ml), 2 equivalentsof DIPEA (0.72 ml, 4.14 mmol) and 2.2 equivalents of ethane-1,2-diamine(0.31 ml, 4.56 mmol). The product was recrystallized from ethanol as ayellow solid. Yield 43% (0.27 g), mp 96° C.

¹H NMR (500 MHz, DMSO-d6) δ 12.51 (s, 1H, NH), 11.62 (s, 2H, NH₂), 8.80(s, 1H, NH), 8.60 (dd, J=9.4, 1.1 Hz, 1H, ArH), 8.03 (dd, J=9.4, 1.6 Hz,1H, ArH), 7.98 (dd, J=9.7, 1.6 Hz, 2H, ArH), 7.72-7.64 (m, 2H, ArH),7.62 (td, J=16.6, 1.9 Hz, 1H, ArH), 7.26 (td, J=16.4, 1.1 Hz, 1H, ArH),3.91 (s, 4H, CH₂).

¹³C NMR (126 MHz, DMSO-d6) δ 168.24 (C═O), 160.87 (d, J_(C-F)=151.5 Hz,ArC—F), 158.29 (C═O), 154.31 (ArC), 138.41 (ArC), 133.74 (d, J_(C-F)=9.5Hz, ArCH), 131.80 (ArCH), 130.61 (d, J_(C-F)=1.8 Hz, ArCH), 128.21(ArCH), 125.02 (d, J_(C-F)=3.6 Hz, ArCH), 123.22 (ArCH), 121.65 (ArC),120.86 (ArCH), 116.57 (d, J_(C-F)=22.5 Hz, ArCH), 42.79 (CH₂), 40.88(CH₂).

MS (EI+): 301.12. High resolution MS: 302.1301 [M−1].

Composition: C21H1802 (delta ppm −0.1), C13H1902F5 (delta ppm 0.4),C16H17O2N3F1 (delta ppm 0.6).

Example 3 Synthesis of Compounds of Series 3

The compounds of series 3 have the general formula

Compound R⁴ 3a 3-OCH₃ 3b 3-CH₃ 3c 5-I 3d 6-Cl

Step 1—Synthesis of Intermediates Synthesis of2-(2-fluorophenyl)-8-methoxy-3,1-benzoxazin-4-one (Intermediate 3a)

Chemical Formula: C15H1FNO3 Molecular Weight: 271.24

The synthetic procedure followed the general method set out above forstep 1 using 2-amino-3-methoxybenzoic acid (0.50 g, 2.99 mmol) dissolvedin pyridine (5 ml) and 2.2 equivalent of 2-fluorobenzoyl chloride (0.79ml, 6.58 mmol). Collected as a white solid, yield 92% (0.75 g), mp 131°C.

¹H NMR (500 MHz, DMSO-d6) δ 8.08 (td, J=7.7, 1.8 Hz, 1H, ArH), 7.72 (d,J=1.6 Hz, 1H, ArH), 7.71 (d, J=1.6 Hz, 1H, ArH), 7.63-7.54 (m, 2H, ArH),7.42 (m, 2H, ArH), 3.97 (s, 3H, OCH₃).

13C NMR (126 MHz, DMSO-d6) δ 160.31 (d, J_(C-F)=257.0 Hz, ArC—F), 158.73(ArC═O), 154.15 (ArC), 152.92 (ArC), 135.83 (ArC), 134.31 (d,J_(C-F)=8.8 Hz, ArCH), 131.14 (ArCH), 129.48 (ArCH), 124.79 (d,J_(C-F)=3.8 Hz, ArCH), 118.90 (d, J_(C-F)=10.1 Hz, ArC), 118.78 (ArCH),118.32 (ArCH), 117.22 (d, J_(C-F)=21.4 Hz, ArCH), 56.34 (OCH3).

MS (APCI+): 272.07 [M+1].

Synthesis of 2-(2-fluorophenyl)-8-methoxy-3,1-benzoxazin-4-one(Intermediate 3b)

Chemical Formula: C15H10FNO2 Molecular Weight: 255.21

The synthetic procedure followed the general method set out above forstep 1 using 2-amino-3-methylbenzoic acid (0.50 g, 3.31 mmol) dissolvedin pyridine (5 ml) and 2.2 equivalent of 2-fluorobenzoyl chloride (0.87ml, 7.28 mmol). Collected as a white solid, yield 97% (0.81 g), mp 106°C.

¹H NMR (500 MHz, DMSO-d6) δ 8.13 (td, J=7.7, 1.9 Hz, 1H, ArH), 8.0 (d,J=8.8 Hz, 1H, ArH), 7.84 (d, J=8.8, 1 H, ArH), 7.75-7.67 (m, 1H, ArH),7.55 (t, J=7.7 Hz, 1H, ArH), 7.47-7.40 (m, 2H, ArH), 2.58 (s, 3H, CH₃).

¹³C NMR (126 MHz, DMSO-d6) δ 160.51 (d, J_(C-F)=259.6 Hz, ArC—F), 159.01(ArC═O), 153.06 (ArC), 144.29 (ArC), 137.40 (ArCH), 135.61 (ArC), 134.44(d, J_(C-F)=8.82 Hz, ArCH), 131.07 (ArCH), 128.42 (ArCH), 125.54 (ArCH),124.82 (d, J_(C-F)=3.8 Hz, ArCH), 118.93 (d, J_(C-F)=1.3 Hz, ArC),117.32 (d, J_(C-F)=21.4 Hz, ArC), 116.78 (ArCH), 16.50 (CH₃).

MS (APCI+): 256.07 [M+1].

Synthesis of 2-(2-fluorophenyl)-8-methoxy-3,1-benzoxazin-4-one(Intermediate 3c)

Chemical Formula: C14H7FINO2 Molecular Weight: 367.11

The synthetic procedure followed the general method set out above forstep 1 using 2-amino-5-iodobenzoic acid (0.50 g, 1.90 mmol) dissolved inpyridine (5 ml) and 2.2 equivalent of 2-fluorobenzoyl chloride (0.50 ml,4.18 mmol). Collected as a white solid, yield 90% (0.63 g), mp 159° C.

¹H NMR (500 MHz, DMSO-d6) δ 8.41 (d, J=2.0 Hz, 1H, ArH), 8.26 (dd,J=8.4, 2.0 Hz, 1H, ArH), 8.09 (td, J=7.8, 1.8 Hz, 1H, ArH), 7.75-7.69(m, 1H, ArH), 7.50 (d, J=8.4 Hz, 1H, ArH), 7.44 (m, 2H, ArH).

¹³C NMR (126 MHz, DMSO-d6) δ 160.48 (d, J_(C-F)=258.3 Hz, ArC—F), 157.35(ArC═O), 154.59 (ArC), 145.39 (ArC), 145.12 (ArCH), 135.85 (ArCH),134.74 (d, J_(C-F)=8.8 Hz, ArCH), 131.10 (ArCH), 128.95 (ArCH), 124.86(d, J_(C-F)=3.8 Hz, ArCH), 118.82 (ArC), 118.45 (d, J_(C-F)=8.8 Hz,ArC), 117.28 (d, J_(C-F)=21.4 Hz, ArCH), 93.91 (ArC—I).

Synthesis of 5-chloro-2-(2-fluorophenyl)-3,1-benzoxazin-4-one(Intermediate 3d)

Chemical Formula: C14H7CIFNO2 Molecular Weight: 275.66

The synthetic procedure followed the general method set out above forstep 1 using 2-amino-6-chlorobenzoic acid (0.50 g, 2.91 mmol) dissolvedin pyridine (5 ml) and 2.2 equivalent of 2-fluorobenzoyl chloride (0.76ml, 6.41 mmol). Collected as a white solid, yield 89% (0.71 g), mp 104°C.

1H NMR (500 MHz, DMSO-d6) δ 8.01 (td, J=17.6, 1.8 Hz, 1H, ArH), 7.90 (t,J=16.1 Hz, 1H, ArH), 7.71 (dd, J=9.1, 1.2 Hz, 2H, ArH), 7.67 (dd, J=9.1,1.2 Hz, 1H, ArH), 7.46-7.42 (m, 2H, ArH),

¹³C NMR (126 MHz, DMSO-d6) δ 160.53 (d, J_(C-F)=256.9 Hz, ArC—F), 155.43(ArC═O), 148.53 (ArC), 144.39 (ArC), 136.6 (ArCH), 134.81 (d,J_(C-F)=10.1 Hz, ArCH), 134.01 (ArC), 131.09 (ArCH), 130.93 (ArCH),126.44 (ArCH), 124.86 (d, J_(C-F)=3.9 Hz, ArCH), 118.18 (d, J_(C-F)=10.3Hz, ArC), 117.28 (d, J_(C-F)=22.3 Hz, ArCH), 114.77 (ArC—Cl).

MS (EI+): 275.01.

Step 2—Synthesis of Example Compounds Synthesis of2-[(2-fluorobenzoyl)amino]-3-methoxy-N-(2-morpholin-4-ylethyl) benzamide(Compound 3a)

Chemical Formula: C21H24FN3O4 Molecular Weight: 401.43

The synthetic procedure followed the general method for step 2 usingIntermediate 3a (0.50 g, 1.84 mmol) in DMF (8 ml), 2 equivalents ofDIPEA (0.64 ml, 3.69 mmol) and 2.2 equivalents of 2-morpholinoethanamine(0.53 ml, 4.06 mmol). The product was recrystallized from ethanol as awhite solid. Yield 14% (0.27 g), mp 131° C.

¹H NMR (500 MHz, DMSO-d6) δ 9.53 (s, 1H, NH), 8.02 (s, 1H, NH), 7.79 (t,J=15.1 Hz, 1H, ArH), 7.60 (q, J=7.0 Hz, 1H, ArH), 7.38-7.30 (m, 3H,ArH), 7.21 (d, J=8.2 Hz, 1H, ArH), 7.12 (d, J=7.7 Hz, 1H, ArH), 3.81 (s,3H, OCH₃), 3.31 (t, J=10.1 Hz, 6H, CH₂), 2.33 (m, 6H, CH₂).

¹³C NMR (126 MHz, DMSO-d6) δ 166.71 (C═O), 159.56 (d, J_(C-F)=249.5 Hz,ArC—F), 154.59 (C═O), 144.51 (ArC), 138.35 (ArC), 134.90 (d, J_(C-F)=1.3Hz, ArC), 133.09 (d, J_(C-F)=1.3 Hz, ArCH), 130.62 (ArCH), 127.27(ArCH), 124.50 (ArCH), 119.74 (ArCH), 118.6 (ArC), 116.34 (d,J_(C-F)=22.7 Hz, ArCH), 113.45 (ArCH), 66.05 (CH₂), 57.03 (CH₂), 56.04(OCH₃), 53.14 (CH₂), 36.23 (CH₂).

MS (ES+): 402.21 [M+1].

Calculated analysis for C21H24FN3O4 (401.43): C, 62.83; H, 6.03; N,10.47. Found C, 62.76; H, 6.17; N, 10.53.

Synthesis of2-[(2-fluorobenzoyl)amino]-3-methyl-N-(2-morpholin-4-ylethyl) benzamide(Compound 3b)

Chemical Formula: C21H24FN3O3, Molecular Weight: 385.43

The synthetic procedure followed the general method for Step 2 using2-(2-fluorophenyl)-8-methyl-3,1-benzoxazin-4-one (0.50 g, 1.96 mmol) inDMF (8 ml), 2 equivalents of DIPEA (0.68 ml, 3.91 mmol) and 2.2equivalents of 2-morpholinoethanamine (0.57 ml, 4.31 mmol). The productwas recrystallized from ethanol as a white solid. Yield 42% (0.32 g), mp165° C.

¹H NMR (500 MHz, DMSO-d6) δ 9.96 (s, 1H, NH), 8.12 (s, 1H, NH), 7.80(td, J=7.6, 1.9 Hz, 1H, ArH), 7.61 (td, J=7.4, 1.9 Hz, 1H, ArH), 7.37(m, 4H, ArH), 7.30 (t, J=7.6 Hz, 1H, ArH), 3.32 (d, J=5.4 Hz, 4H, CH₂),2.39 (t, J=6.8 Hz, 2H, CH₂), 2.32 (m, 6H, CH₂), 2.27 (s, 3H, CH₃).

¹³C NMR (126 MHz, DMSO-d6) δ 167.43 (C═O), 162.08 (d, J_(C-F)=206.6 Hz,ArC—F), 158.45 (C═O), 135.89 (ArC), 133.90 (ArC), 133.42 (ArC), 133.04(d, J_(C-F)=8.8 Hz, ArCH), 131.88 (ArCH), 130.36 (d, J_(C-F)=1.3 Hz,ArCH), 126.37 (ArCH), 125.59 (ArCH), 125.10 (ArCH), 124.62 (d,J_(C-F)=2.5 Hz, ArC), 116.30 (d, J_(C-F)=22.7 Hz, ArCH), 66.08 (CH₂),57.04 (CH₂), 53.16 (CH₂), 36.30 (CH₂), 18.22 (CH₃).

MS (ES+): 386.22 [M+1].

Calculated analysis for C21H24FN3O3 (385.43): C, 65.44; H, 6.28; N,10.90. Found C, 65.66; H, 6.27; N, 10.96.

Synthesis of2-[(2-fluorobenzoyl)amino]-2-chloro-N-(2-morpholin-4-ylethyl) benzamide(Compound 3d)

Chemical Formula: C20H21 FCIN3O3 Molecular Weight: 405.85 The syntheticprocedure followed the general method for Step 2 using2-(2-fluorophenyl)-5-chloro-3,1-benzoxazin-4-one (0.50 g, 1.83 mmol) inDMF (8 ml), 2 equivalents of DIPEA (0.64 ml, 3.65 mmol) and 2.2equivalents of 2-morpholinoethanamine (0.53 ml, 4.02 mmol). The productwas recrystallized from ethanol as a yellow solid. Yield 33% (0.25 g),mp 121° C.

¹H NMR (500 MHz, DMSO-d6) δ 9.60 (s, 1H, NH), 8.60 (s, 1H, NH), 7.97(dd, J=8.6 Hz, 1H, ArH), 7.87 (td, J=15.6, 1.8 Hz, 1H, ArH), 7.67-7.63(m, 1H, ArH), 7.48 (t, 16.5 Hz, 1H, ArH), 7.42-7.36 (m, 3H, ArH), 3.48(t, J=9.3 Hz, 4H, CH₂), 3.38 (q, J=19.5 Hz, 2H, CH₂), 2.43 (t, J=13.7Hz, 2H, CH₂), 2.33 (s, 4H, CH₂).

¹³C NMR (126 MHz, DMSO-d6) δ 164.21 (C═O), 162.52 (d, J_(C-F)=114.8 Hz,ArC—F), 158.45 (C═O), 155.98 (ArC), 142.8 (ArC), 136.0 (ArC), 133.97 (d,J_(C-F)=9.4 Hz, ArCH), 130.87 (ArCH), 130.24 (ArCH, 130.20 (d,J_(C-F)=19.9 Hz, ArC), 125.94 (ArCH), 124.99 (d, J_(C-F)=3.4 Hz, ArCH),122.60 (ArCH), 116.44 (d, J_(C-F)=23.0 Hz, ArCH), 66.08 (CH₂), 56.74(CH₂), 53.16 (CH₂), 36.47 (CH₂).

MS (EI+): 405.12.

Calculated analysis for C20H21FCIN3O3 (405.85): C, 59.19; H, 5.22; N,10.35. Found C, 59.40; H, 5.21; N, 10.38.

BIOLOGICAL EXAMPLES Materials and Methods

Cloning Procedures

NF-κB Luciferase Reporter Plasmid

NF-κB luciferase assays were carried out using the 3× κB luciferasereporter plasmid, which was a kind gift from Professor Ron Hay(University of St. Andrews). A pcDNA3.1 plasmid containing the LacZsequence was used as a control for transfection efficiency (gift fromProfessor Trevor Dale, School of Biosciences, Cardiff University). Forpositive and negative controls pGL3 luciferase reporter vectors(Promega) were used, pGL3control and pGL3basic, respectively.

Cell Culture Maintenance and Storage

Experimental Cell Lines

The human embryonic kidney cells (HEK-293) were a gift from Prof.Vladimir Buchman (School of Biosciences, Cardiff University). The humanbreast cancer cell lines, MDA-MB-231, SKBR3 and ZR-7S-1 were a gift fromDr. Julia Gee (Department of Pharmacy and Pharmaceutical Sciences,Cardiff University). The human normal breast cancer cell line MCF-10Awas a gift from Dr. Torsten Stein (Division of Cancer Sciences andMolecular Pathology, University of Glasgow). Descriptions of the maincell lines used are outlined below:

HEK-293 is a non-tumorigenic cell line derived from human embryonickidney cells. HEK-293 cells are convenient for our investigation,because they are easily transfected and have undetectable basal level ofBcl-3 protein.

MDA-MB-231 is a highly metastatic, human basal epithelial cell lineisolated from the pleural effusion of an adenocarcinoma. The cells are‘triple negative’ as they lack estrogen, progesterone and ERBB2 receptorand they strongly over-express EGFR. The expression of receptors in thisline has been confirmed by the host laboratory.

SKBR3 cell line is a poorly metastatic human luminal epithelial cellline derived from a pleural effusion. SKBR3 cells are estrogen andprogesterone receptor negative, over-express the ERBB2 receptor and havevery low levels of the EGFR receptor.

The ZR-7S-1 cell line is a moderately metastatic human luminalepithelial cell line derived from a malignant ascitic effusion withinfiltrating ductal carcinoma. ZR-7S-1 cells are oestrogen andprogesterone receptor positive. They express very low levels of theErbB2 receptor and over-express EGFR.

MCF-10A is a mammary epithelial cell line and is considered as a modelof non-tumorigenic mammary cells. MCF-10 cells were derived from amammary tissue from a 36-year-old woman in a good health and theimmortalized MCF-10A line can grow in culture and has a stable,near-diploid karyotype with modest genetic modifications typical ofculture-adapted breast epithelial cells.

Maintenance of Cell Lines

The HEK-293 cell line was maintained in Dulbecco's modified Eagle'smedium (DMEM, Invitrogen) supplemented with 10% v/v foetal bovine serum(FBS, Sigma, Dorset, UK), penicillin (50 u/ml, Invitrogen), streptomycin(50 u/ml, Invitrogen) and L-glutamine (2 mM, Invitrogen). TheMDA-MB-231, ZR-75-1 and SKBR3 cell lines were maintained in RPMI medium(Invitrogen) supplemented with 10% v/v FBS, penicillin (50 u/mlInvitrogen) streptomycin (50 u/ml, Invitrogen) and L-glutamine (2 mM,Invitrogen). MCF-10A cell line was maintained in Dulbecco's modifiedEagle's medium nutrient mixture F-12 (DMEM/F-12, Invitrogen)supplemented with 5% v/v horse serum (Sigma, Dorset, UK), penicillin (50u/ml, Invitrogen), streptomycin (50 u/ml, Invitrogen), epidermal growthfactor (EGF, 20 ng/ml, Sigma), hydrocortisone (0.5 mg/ml, Sigma),cholera toxin (100 mg/ml, Sigma) and insulin (10 μg/ml, Sigma).

All cell lines were incubated at 37° C. and 5% CO₂ in T25 or T80 tissueculture flasks (Nunc, Leics, UK) and were routinely passaged every 3-8days at a split ratio of 1:4-1:12, when they became 80-90% confluent.

Cell Based Assays

Cell Titre Blue Viability Assay

The viability of cells at experimental endpoints for particular assayswas determined using the Cell Titre Blue reagent (Promega, Southampton,UK). This reagent measures the cellular metabolic activity usingresazurin as an indicator dye. Viable cells, therefore metabolicallyactive, will reduce resazurin into highly fluorescent resofurin. Theresulting fluorescence levels are measured and indicate cell viability.

Cells were plated at low confluency into 96 well plates in 100 μl ofcomplete growth media in triplicates and were incubated at 37° C. in 5%CO₂ for the desired test exposure period. For each 100 μl of media in 96well plates, 20 μl Cell Titre Blue reagent was added followed byincubation for an hour at 37° C. in 5% CO₂. Fluorescence was thenmeasured by setting excitation/emission wavelengths to 560/590 nm on aFlurostar Optima plate reader (BMG tabtech, Bucks, UK).

Cell Count

To establish cell viability over time period of three days, respectivecells were seeded at low confluency into 96 well plates in 100 μl ofcomplete growth media in triplicates and were incubated at 37° C. in 5%CO₂. After 24 hrs, cells from triplicate wells for the first time pointwere detached using 0.25% Trypsin/EDTA (Invitrogen) and resuspended incomplete growth media and individually counted. The same was done foreach cell line at 48 hrs and 72 hrs post-seeding.

Determination of NF-κB Activity in Cells

For NF-κB luciferase assays, cells were seeded into clear bottom black96-well plates (Corning Inc., Lowell, US) in antibiotic free culturemedia in appropriate density. After 24 hrs, cells were transfected with10 ng of 3× κB luciferase plasmid and 10 ng of pcDNA3.1-Lacl plasmid perwell. Empty pcDNA3.1 plasmid was also included to normalize the totalweight of DNA transfected to 100 ng. For positive and negative controlsrespectively, 10 ng of pGL3control or pGL3basic were transfected inplace of 3× κB luciferase plasmid. Transfection was carried out usingLipofectamine LTX reagents (Invitrogen, Paisley, UK).

After 48 hrs post-transfection with luciferase reporter plasmid, themedia was aspirated and cells were lyzed using 50 μl/well of Glo-lysisbuffer (Promega, Southampton, UK). The plate was left on a rocker for 20min to facilitate complete cell lysis. Then, 20 μl of lysate from eachwell was removed and transferred into a new clear bottom black wellplate for measuring LacZ activity as a transfection efficiency controland followed by addition of 20 ul/well of Beta-Glo substrate (Promega,Southampton, UK) and cultivation at room temperature for at least 20min. Subsequently, 30 μl/well of Bright-Glo luciferase substrate(Promega, Southampton, UK) was added to the original plate and assesimmediately for luminescence activity. The luminescence produced fromeither reaction was read using a Flurostar Optima plate reader (BMGtabtech, Bucks, UK). The resulting luciferase activity was thennormalized against lacZ activity obtained from Beta-glo measurement andis displayed as relative light units (R.t.U).

Boyden Chamber Migration Assay

The migratory or invasive capacity of human mammary cancer cell lineswas assessed by the Boyden chamber assay. Cells were seeded in low serummedia in a chamber with porous membrane (transparent polyethyleneterephthalate (PET) membranes with 8 μm pores) as a solid support formotility assays or with a porous membrane coated with Matrigel BasementMembrane Matrix (BD BioCoat Growth factor reduced invasion chambers) forinvasion assay. The cell insert was placed into a well with completegrowth media, therefore cells are stimulated by a serum gradient tomigrate or invade across the membrane through pores.

Total of 750 μl of complete growth media containing 10% of serum wasadded to appropriate wells of a 24 well cell culture insert companionplate (BD Biosciences, Oxford, UK). A cell culture insert (BDBiosciences, Oxford, UK) was then carefully placed into each well of theinsert companion plate using tweezers. Cells were detached from tissueculture plates using 0.25% w/v Trypsin/EDTA (Invitrogen) and centrifugedat 13000 rpm for 5 min. Cells were washed twice in serum free media byresuspension and centrifugation. The appropriate number of cells (2×10⁵cells/ml for MDA-MB-231 cells) was resuspended in normal growth mediacontaining only 0.1% serum. 350 μl of the cell suspension was added tothe appropriate upper chambers of the cell culture inserts and plateswere incubated for 24 hrs at 37° C. and 5% CO₂.

After incubation, cells on membranes were fixed by replacing the mediain the top and bottom sections of the chamber with 70% ice-cold ethanol(Fisher Scientific). Plates were incubated at −20° C. for at least anhour. After fixation, inserts were immersed in a tap water usingtweezers to ensure all ethanol was removed. A moistened cotton wool budwas then used to mechanically remove all cells fixed on the upper sideof the membranes. Cells were stained by individually immersing theinserts into filtered Harris' Haematoxylin (Sigma, Dorset, UK) for 1min. Following this, inserts were washed in a beaker of tap water toremove the dye and immersed in 0.5% filtered Eosin (Sigma, Dorset, UK)for 2 min. Stained inserts were then washed again in a tap water.

Glycerol Gelatin (Sigma, Dorset, UK) was heated in a beaker of boilingwater and once liquefied, a drop was placed onto an appropriatelylabelled microscope slide (R. A. Lamb, Loughborough, UK). Membranes werecut out of the insert and transferred onto the corresponding slide withtweezers. Glycerol Gelatin was added to the top of the membranes and acover slip was placed on the slide under firm pressure. Mounted slideswere left to air dry before being analysed.

Protein Analysis

Protein Extraction from Cells

Proteins were extracted from cells in order to be analyzed by ELISAassay. The media from tissue culture flask was removed and cells wererinsed with ice cold PBS (Sigma, Dorset, UK). Appropriate volume of PBS(5 ml for T25, 10 ml for T80) was added into the flask and cells wereremoved with a cell scraper (Nunc, Leics, UK). The cell suspension wasthen transferred to 15 ml tubes and centrifuged at 11000 rpm for 5 minat room temperature. Resulting pellet was used for protein extractionimmediately or stored at −20° C. prior use.

Non-denatured protein extract was prepared using non-denaturing lysisbuffer (Table 1) and used to analyze protein-protein interaction.

TABLE 1 Composition of buffers for whole cell protein extraction RIPAbuffer pH 7.4 Non-denaturing buffer 50 mM Tris pH 8 (Sigma) 20 mM TrispH 7.5 (Sigma) 150 mM sodium chloride (Sigma) 150 mM sodium chloride(Sigma) 1% v/v Nonidet-P40 (Roche) 1% v/v Nonidet-P40 (Roche) 0.1% w/vsodium dodecyl sulphate 1 mM EDTA pH 8.0 (Fischer (SDS, Sigma)Scientific) 0.5% w/v sodium 1 mM EGTA pH 8.0 deoxycholate (Sigma) (FlukaBiochemika)

Complete mini protease inhibitor tablets (Roche, Welwyn Garden City,UK), 10 mM sodium fluoride (Fluka Biochemika), 1 mM sodium pyrophospateand 1 mM sodium orthovanadate (Sigma, Dorset, UK) were added to thebuffer prior use. Cell pellets were resuspended in appropriate volume ofnon-denaturing buffer (50-200 μl) and cultivated on ice 5 min. Cellsuspensions were transferred to microcentrifuge tubes and sonicated onice (3 times 5s) and centrifuged at 10000 rpm for 10 min at 4° C. Theresultant supernatant was used immediately or stored at −20° C. untilrequired.

ELISA Assay

In our case, ELISA assay was used to detect either the amount ofFlag-Bcl-3 protein alone or Flag-Bcl-3 in complex with p50, usingindirect or sandwich ELISA respectively.

For Indirect and sandwich ELISA assay, Non-denaturing cell lysate(above) was diluted with TBS/T [tris buffer saline (TBS, Calbiochem,Merck) supplemented with 0.5% v/v Tween (Sigma, Dorset, UK)] to aconcentration of 0.5-1 μg/μl and 100 μl was added onto ANTI-Flag coatedflat bottom ELISA plates (Sigma, Dorset, UK). Samples were added intriplicates, while TBS/T was used as a negative control. The plate wascultivated at 37° C. for an hour followed by 3×200 μl washes with TBS/T.Primary antibodies, either Bcl-3 (Santa Cruz Biotech) for indirect ELISAand p50 for sandwich ELISA (Abcam) were added in known volumes (125 μl)and concentrations to each well, cultivated covered from light for anhour at room temperature. Another three 200 μl washes with TBS/T wereperformed before cultivation with alkaline phospatase (AP) conjugatedsecondary antibody. Meanwhile, para-nitrophenylphosphate solution (pNPP,Santa Cruz Biotechnology, Calif., USA) was prepared according tomanufacturer's instructions (5 mg of pNPP disodium salt in 5 ml of pNPPsubstrate buffer). The solution was mixed well and covered from lightprior to use. pNPP is a substrate of choice for use with alkalinephosphatase and produces a soluble end product that is yellow in colour.Therefore colour changes can be measured and represent the amount of APpresent. After cultivation with secondary antibody, the wells werewashed 3×200 μl. TBS/T, pNPP solution was added (50 μl/well) andcultivated for an hour covered from light at room temperature. Thereaction was stopped by addition of 3N NaOH (20 μl/well) and thecolorimetric changes were measured at 405 nm using a plate reader.

Statistical Analysis

The Student's T-test was used to determine statistical differencesbetween normally distributed data sets and between data sets with samplesizes of n=3. This test was performed using Excel 2008 software.

Example 4 Characterization of Example Compound 1a

We established that the solubility of Compound 1a in most commonly usedsolvents is very low (Table 2) which represents an issue for biologicalevaluation. Therefore a hydrochloric salt of Compound 1a was synthesisedand the solubility was analysed. The obtained salt had improvedsolubility in water and methanol (Table 2), however it was not soluble(<0.1 g/100 ml) in Phosphate Buffered Saline (PBS). PBS is a water-basedsalt solution containing sodium chloride, sodium phosphate, potassiumchloride and potassium phosphate, while the buffer's phosphate groupshelp to maintain a constant pH. PBS is non-toxic and is commonly used asan isotonic buffered solution for cell-based assays and animal studies.

TABLE 2 Solubility of the lead compound in organic solvents Solubility(g/100 ml) Compound 1a Solvent Compound 1a hydrochloride salt Water<0.01 0.95 Methanol 0.20 0.59 Ethanol 0.09 0.19 DMSO 3.71 <0.01 Ethylacetate 0.34 0.02 Dichloromethane 2.91 0.90 Diethyl ether 0.09 0.01

Example 5 Cell Toxicity of Compound 1a in Vitro

The toxicity of Compound 1a was evaluated in vitro using human breastcancer cell lines. To compare the toxicity in tumorigenic as well as innon-tumorigenic breast cancer cells, we selected MCF-10A as anon-tumorigenic human breast cancer cell line and MDA-MB-231 and SKBR3as cell models of tumorigenic human breast cancer cell line.

MCF-10 cells were derived from a mammary tissue from a 36-year-old womanin a good health and the immortalized MCF-10A line can grow in cultureand has a stable, near-diploid karyotype with modest geneticmodifications typical of culture-adapted breast epithelial cells,including loss of p16 locus. The cells express normal p53 and they donot grow in immuno-compromised mice.

Compound 1a was dissolved in DMSO and diluted in media in a highestconcentration of 1 mM (10⁻³M). Cell toxicity was evaluated using theCell Titre Blue viability assay over a range of molarities for 24 hrs.The effect of Compound 1a on cell viability was always normalisedagainst DMSO control and the dose-response curve was generated usingGraphPad software (FIG. 1).

IC₅₀ values could not be established in any of the cell lines, as eventhe highest concentration did not cause a 50% decrease in cellviability. We could, however, see a difference in cell toxicity betweennon-tumorigenic and tumorigenic cell lines. At the highest concentrationof 1 mM, the viability in MCF-10A cell line was 96%, 72% in MDA-MB-231and 57% in SKBR3 compared to DMSO control (100%). This low toxicity wasexpected of a specific inhibitor of Bcl-3 as previous studies had shownthat genetic inhibition of Bcl-3 had only a modest effect on cellviability of cancer cell lines in vitro and little or no effect onnon-tumourgenic lines (11).

The IC₅₀ was calculated using GraphPad software by extrapolating thedose-response curve, giving IC₅₀ values of 14.9 mM for MCF-10A, 2.70 mMfor MDA-MB-231 and 1.37 mM for SKBR3.

Example 6 Establishing Biological Effects of Compound 1a in Vitro

A. Establishing Effect on Protein Binding by Indirect Sandwich ELISA

HEK-293 cells overexpressing Bcl-3 were cultivated with Compound 1a overa range of molarities for 24 hrs before cell lysates were obtained undernon-denaturing conditions. The dose response curve was generated usingGraphPad software (FIG. 2A). The determined IC₅₀ was 385.5 nM. IndirectELISA assay was performed using Bcl-3 antibody to show an equal loadingacross samples treated with compound 1a (FIG. 2B) and controls (FIG.2C).

B. Establishing the Effect on Intra-Cellular NF-κB Activity.

The effect of Compound 1a on NF-κB activity was determined by NF-κBluciferase assay in MDA-MB-231 and HEK-293 cells overexpressing Bcl-3and HEK-293 cells overexpressing p52. Cells were cultivated withCompound 1a over a range of molarities for 24 hrs before beingtransfected with NF-κB luciferase reporter plasmid for 48 hrs andanalysed for NF-κB activity. The dose response curve was generated usingGraphPad software (FIG. 3). The determined IC₅₀ in MDA-MB-231 cells was49.43 nM, 159.6 nM HEK-293 cells and 210.6 nM in HEK-293 p52overexpressing cells.

C. Establishing the Effect of Compound 1a on Cell Motility.

It was previously determined that Compound 1a significantly suppressedmigration ability in MDA-MB-231 cells at 10 μM. We therefore generated adose response curve to determine an IC₅₀ in this assay. MDA-MB-231 cellsoverexpressing Bcl-3 were cultivated with Compound 1a and DMSO controlover a range of molarities for 24 hrs before being seeded onto Boydenmigration chambers. The constant number of live cells present duringthis assay across samples was monitored by cell count. The dose responsecurve was generated using GraphPad software (FIG. 4). The determinedIC₅₀ was 310.4 nM.

Example 7 Biological Evaluation of Analogues from Series 1-3

A. Cell Toxicity

Selected compounds were dissolved in DMSO and diluted to a highestconcentration of 10 μM (0.1% DMSO). In all assays, a DMSO control wasalways used. Selected compounds were tested for cell toxicity in HEK-293and MDA-MB-231 cells before being used in cell-based assays. Celltoxicity was evaluated using the Cell Titre Blue viability assay over arange of molarities for 24 hrs

Results for mono-substituted compounds 1a, 1c, 1e, 1f, 1 g, 1i, 1j, 2a,2b, 2c, 2d, 2f, 3a, 3b and 3d are shown in FIG. 5. Compounds were welltolerated in both cell lines and cell viability was above 70% even at 10μM concentration.

Results for di- and tri-substituted compounds 1l, 1m, 1n, 1o, 1p and 1qare shown in FIGS. 11A&B. Compounds were well tolerated in both celllines and cell viability was above 90% even at 10 μM concentration.

B. NF-κB Assay

The effect of selected analogues on NF-κB activity was determined byNF-KB luciferase assay in MDA-MB-231 cells. MDA-MB-231 Bcl-3over-expressing cells were cultivated with compounds from series 1-3 at1 μM concentration for 24 hrs before being transfected with NF-κBluciferase reporter plasmid for 48 hrs together with controls andanalysed for NF-κB activity.

Results for mono-substituted compounds 1a, 1c, 1e, 1f, 1 g, 1i, 1j, 2a,2b, 2c, 2d, 2f, 3a, 3b and 3d are shown in FIG. 6).

It can be seen that the NF-κB activity of the mono-substituted analoguesfrom series 1 was comparable to that of Compound 1a, with a significantdecrease of in NF-κB activity observed for Compound 1a and the analogue1f as compared to DMSO control.

From series 2 analogue 2a and 2d significantly decreased NF-κB activityas compared to Bcl-3 WT DMSO control. Analogue 2a showed comparableactivity with Compound 1a with other analogues having lesser activity.

From series 3 analogue 3a had comparable effect on NF-κB activity withCompound 1a. Analogue 3c also showed significant decrease in NF-κBactivity as compared to DMSO control, however not to the same level asCompound 1a.

Based on the results for the series of analogues, we have establisheddose response curve for selected analogues from series 1 (10, fromseries 2 (2a and 2c) and from series 3 (3a) over a range of molarities.MDA-MB-231 Bcl-3 WT cells were cultivated with selected compounds fromseries 1-3 over a range of molarities for 24 hrs before beingtransfected with NF-κB luciferase reporter plasmid for 48 hrs togetherwith controls and analysed for NF-κB activity (FIG. 7). We observed animprovement in the determined IC₅₀ for analogue 1f (6.97 nM). Otheranalogues showed decreased ability to suppress NF-κB activity comparedwith Compound 1a. The determined IC₅₀ for analogue 2a was 2.50 μM, 2.49μM for analogue 2c and 1.07 μM for analogue 3a (see Table 3).

Results for di- and tri-substituted compounds 1l, 1m, 1n, 1o, 1p and 1qare shown in FIG. 11C (compared with Compound 1a) and FIG. 12 as well asin Table 3.

Compound 1a showed the most potent suppression of NF-κB activity ascompared to Bcl-3 WT overexpressing MDA-MB-231 cells (FIG. 11C).

IC₅₀ values were established for three selected analogues (1o-q). Allthree tested analogues showed decreased ability to suppress NF-κBactivity than Compound 1a. The determined IC₅₀ for analogue 10 was237.80 nM, 705.90 nM for analogue 1p and 988.87 nM for analogue 1q (FIG.12; Table 3).

TABLE 3 Comparison of IC₅₀ values for test compounds ELISA NF-κB Cellmotility Analogue assay (μM) assay (μM) assay (μM) 1a 0.3855 0.045430.3104 1f 0.0617 0.00697 0.02893 2a 15.23 2.5 1.33 2c 10.41 2.49 3.65 3a0.01195 1.07 0.90 1o 0.2378 1p 0.7059 1q 0.98887C. Indirect Sandwich ELISA Assay

The ability to disrupt Bcl-3-p50 binding was determined by IndirectSandwich ELISA assay for selected analogues (1f, 2a, 2c, 3a).

HEK-293 Bcl-3 WT cells were cultivated with selected compounds over arange of molarities for 24 hrs before cell lysates were obtained undernon-denaturing conditions. The dose response curve for selectedanalogues was generated using GraphPad software (FIG. 8A-D). We observedimproved IC50 as compared to Compound 1a for analogues 1f and 3a, withIC50 values 60.17 nM and 119.3 nM respectively. The IC50 could not beestablished for analogues from series 2, 2a and 2c, and was calculatedfrom the dose response curve using GraphPad software with IC₅₀ of 15.23μM for analogue 2a and 10.41 μM for analogue 2c.

Indirect ELISA assay was performed using Bcl-3 antibody to show an equalloading across samples treated with compounds 1f, 2a, 2c, and 3a (FIG.8E-H).

D. Cell Motility Assay

As shown in Example 6, Compound 1a caused a significant decrease in cellmotility with an IC₅₀ value of 310.4 nM. Therefore we wanted toestablish whether designed analogues have similar or improved ability tosuppress cell migration. MDA-MB-231 Bcl-3 over-expressing cells werecultivated with selected analogues (1f, 2a, 2c, 3a) and DMSO controlover a range of molarities for 24 hrs before being seeded onto theBoyden motility chambers. Migrated cells were visualised and countedafter 24 hrs. The dose response curves were generated using GraphPadsoftware (FIG. 9). The constant number of live cells present during thisassay across samples was monitored by cell count.

Consistent with results from NF-κB assay and Indirect Sandwich ELISAassay, the analogue 1f showed an improved ability to suppress cellmigration, with IC50 value of 28.93 nM. Interestingly, the cellmigration was suppressed below 50% even at 10 nM concentration. Otheranalogues showed decreased ability to suppress cell motility thanCompound 1a. The determined IC50 for analogue 2a was 1.33 μM, 3.65 μMfor analogue 2c and 900 nM for analogue 3a.

F. Establishing EC₅₀ of Analogue 1f

The activity of the analogue 1f was improved as compared to Compound 1a;therefore next we determined the toxicity of this analogue in MDA-MB-231cell line. Compound 1a was dissolved in DMSO and diluted in media to ahighest concentration of 2 mM. Cell toxicity was evaluated using theCell Titre Blue viability assay over a range of molarities for 24 hrs.The effect of Compound 1a on cell viability was always normalisedagainst DMSO control and the dose-response curve was generated usingGraphPad software (FIG. 10). The determined EC₅₀ for analogue 1f was781.3 μM.

SUMMARY

We have synthesised2-[(2-fluorobenzoyl)amino]-N-(2-morpholin-4-ylethyl)benzamide (Compound1a) and a number of novel analogues of this compound and we have shownthat the compounds are inhibitors of Bcl3 since are capable ofsuppressing Bcl3-NFkB protein interactions and inhibiting NF-κBsignalling. This indicates that the compounds will be useful for thetreatment of cancer, particularly metastatic cancer.

REFERENCES

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The invention claimed is:
 1. A compound selected from:2-[(2-methoxybenzoyl)amino]-N-(2-morpholin-4-ylethyl)benzamide;2-[(3-methoxybenzoyl)amino]-N-(2-morpholin-4-ylethyl)benzamide;2-[(4-nitrobenzoyl)amino]-N-(2-morpholin-4-ylethyl)benzamide;2-[(2-methylbenzoyl)amino]-N-(2-morpholin-4-ylethyl)benzamide;3,5-difluoro-N-(2-[(2-morpholin-4-ylethyl)carbamoyl]phenyl)benzamide;2,6-difluoro-N-(2-[(2-morpholin-4-ylethyl)carbamoyl]phenyl)benzamide;2,4-difluoro-N-(2-[(2-morpholin-4-ylethyl)carbamoyl]phenyl)benzamide;2-[(4-fluorobenzoyl)amino]-N-(pyrrolidin-3-ylmethyl)benzamide;2-[(4-fluorobenzoyl)amino]-N-(piperidin-3-ylmethyl)benzamide;2-[(4-fluorobenzoyl)amino]-N-(piperazin-3-ylmethyl)benzamide;N-(2-aminoethyl)-2-(2-fluorobenzamido)benzamide;2-[(2-fluorobenzoyl)amino]-3-methyl-N-(2-morpholin-4-ylethyl)benzamide;and2-[(2-fluorobenzoyl)amino]-3-methoxy-N-(2-morpholin-4-ylethyl)benzamide;or a pharmaceutically or veterinarily acceptable salt thereof.